EA030795B1 - Molecules for selective delivery of a pair of imaging agents to a cancerous tissue - Google Patents

Abstract

Disclosed herein are a molecule for selective delivery of a pair of agents for visualizing a cancerous tissue and visualizing a cancerous tissue, having the structure D-c]-A-[c-M]-X-B-[c-D], wherein X is a peptide linker cleavable by a protease; A is a peptide with a sequence comprising 5 consecutive glutamate residues; B is a peptide with a sequence comprising 8 consecutive arginine residues; C, Cand Care each independently an amino acid residue; M is a polyethylene glycol (PEG) polymer; and Dand Dare a pair of acceptor and donor fluorescent moieties that are capable of undergoing Forsters fluorescence resonance energy transfer with the other; and wherein [C-M] is bound to at any position on X, [D-C] is bound to any amino acid on A, and [C-D] is bound to any amino acid on B, and M is a polyethylene glycol (PEG) polymer having an average molecular weight of 500, 1000, 2000, 5000 or 10000 Da, and also the use thereof for delivering a pair of imaging agents to a cancerous tissue.

Description

This invention relates to a molecule for the selective delivery of a pair of cancer tissue imaging agents having the formula I having the structure | T) a-ca] -A- [cm-M] -X-B- [sv-Ov] formula I, where X is a peptide linker that is capable of being cleaved by a protease;

A is a peptide with a sequence containing 5 consecutive glutamate residues;

B is a peptide with a sequence containing 8 consecutive arginine residues;

CA, CB and CM, each independently, is an amino acid residue;

M is a polyethylene glycol polymer (PEG);

1) _A and 1) _B are a pair of acceptor and donor fluorescent particles, between which fluorescence resonant energy transfer is possible according to the Förster mechanism; and where [c _M- M] is attached in any position to X, [E _A -c _A ] is attached to any amino acid in A, and | c _B -1) _B ] is attached to any amino acid in B, M is a polyethylene glycol polymer (PEG) having an average molecular weight of 500, 1000, 2000, 5000 or 10000 Da.

In one embodiment of the invention with _A , _B, and _M , each independently selected from β-cysteine, β-glutamate, lysine and para-4-acetyl-β-phenylalanine. c _B is a β-cysteine residue.

In one embodiment of the invention, _A represents a β-glutamate or lysine residue.

In one embodiment of the invention, _M is the residue of para-4-acetyl-L-phenylalanine.

In one embodiment of the invention, X is capable of cleavage using matrix metalloproteinase.

In one embodiment of the invention, X contains an amino acid sequence that is capable of cleavage using MMP2, MMP7, MMP9 or MMP14. X contains an amino acid sequence selected from

RShAO, RGO-S (she) AO, ΚΡΙΑΓΑΚδ, Ε8ΡΑΥΥΤΑ, ОРК8РЦ РРК8РЦ РТЦ] Л <к and РТЦи <(Ас).

In one embodiment of the invention, X comprises the amino acid sequence P1.01 .AC.

In one embodiment of the invention, X comprises the amino acid sequence of PBC-C (te) AC.

In one embodiment of the invention, X comprises the amino acid sequence of KP1. A. 1. AG8.

In one embodiment of the invention, E _A and E _{B are} Cy5 and Cy7.

- 1 030795

In one embodiment of the invention, Ό _Α and Ό _Β mean Su5 and 1KOe750.

In one embodiment of the invention, Ό _Α and Ό _Β mean Su5 and 1KOe800.

In one embodiment of the invention, Ό _Α and _{Β Β are} Su5 and 1CO.

In one embodiment of the invention, the molecule of formula (I) is

δϋΜ-25.

In one embodiment of the invention, the tissue is breast cancer tissue, colorectal cancer tissue, squamous cell carcinoma tissue, prostate cancer tissue, melanoma tissue, or thyroid cancer tissue.

In one embodiment of the invention, the tissue is breast cancer tissue.

In one embodiment of the invention, the tissue is colorectal cancer tissue.

In one embodiment of the invention, a tissue sample for cancer imaging comprises (a) tissue and (b) a molecule of formula (I).

In one embodiment of the invention, the tissue sample is a sample placed on a slide or slice to determine pathology.

In one embodiment of the invention, the tissue is cancerous tissue.

In one embodiment of the invention, the cancerous tissue is breast cancerous tissue, colorectal cancerous tissue, squamous cell carcinoma tissue, prostate cancerous tissue, melanoma tissue or thyroid cancerous tissue.

In one embodiment of the invention, the cancerous tissue is breast cancerous tissue.

In one embodiment of the invention, the cancerous tissue is cancerous tissue of colorectal cancer.

In one embodiment of the invention, a molecule of formula (I) is used to deliver a pair of imaging agents to the cancerous tissue, including bringing the cancerous tissue into contact with said molecule.

In one embodiment of the invention, the cancerous tissue is breast cancerous tissue, colorectal cancerous tissue, squamous cell carcinoma tissue, prostate cancerous tissue, melanoma tissue or thyroid cancerous tissue.

In one embodiment, the cancerous tissue is breast cancerous tissue.

In one embodiment, the cancerous tissue is cancerous tissue of colorectal cancer.

In one embodiment of the invention, a molecule of formula (I) is used to visualize cancerous tissue in a subject in need thereof, the use of which includes:

ί) administering the indicated molecule to the subject; and ίί) visualizing at least one of the imaging agents.

In one embodiment, the cancerous tissue is breast cancerous tissue, colorectal cancerous tissue, squamous cell carcinoma tissue, prostate cancerous tissue, melanoma tissue or thyroid cancerous tissue.

In one embodiment, the cancerous tissue is breast cancerous tissue.

In one embodiment, the cancerous tissue is cancerous tissue of colorectal cancer.

In one embodiment, the surgical field surrounding the cancerous tissue is reduced.

In one embodiment of the invention, the molecule of the formula is a molecule of the formula (I) 8ΌΜ-25

8ΌΜ-25.

List of figures

In FIG. Figure 1 shows the effects of a molecule for selective delivery (8ΌΜ), shows fluorescence images of 3 different mice that received an 8ΌΜ-6 injection. Images were obtained 2 hours after injection. The tumor tissue and the contralateral tissue used to determine the contrast are shown in the right image. The average contrast for three mice was 1.1.

In FIG. 2 shows ratiometric changes in the fluorescence intensity of molecules for selective delivery. This figure shows the results of the cleavage of 8ΌΜ-9 using 1 nM enzyme ΜΜΡ-2. An increase in the donor fluorescence intensity (left panel) and a decrease in the acceptor fluorescence intensity (right panel) indicates a decrease in RCET (resonance fluorescence energy transfer) after peptide cleavage.

In FIG. Figure 3 shows the enhancement of the fluorescence intensity of the molecules for selective delivery after cleavage using protease. Cleavage of 8ΌΜ-10 was carried out using 1 nM enzyme ΜΜΡ-9 in physiological saline containing a buffer substance. The fluorescence of the Cy5 label increases more than 100-fold after cleavage of the peptide, since the fluorescence quenchers are no longer attached intramolecularly to Cy5 and can no longer quench Cy5 fluorescence.

In FIG. Figure 4 shows an increase in the fluorescence intensity of the molecule for the selective delivery of 8-10 after cleavage of tumor homogenates. The molecule for the selective delivery of 10 (8ΌΜ-10) was cleaved by the tumor homogenates of NT-1080. 1 nM ΜΜΡ -9 or 10 μl of tumor tissue homogenates (TH2 and TH3) was mixed with 1 μM of compound 13 in 100 μl of buffer solution for 24 hours at 37 ° C. ΟΜ6001 is a general broad-spectrum ΜΜΡδ inhibitor.

The control band contains 8ΌΜ-6, which, in an intact, not cleaved form, is characterized by strong fluorescence, which is manifested on the surface of the gel. The non-cleaved 8ΌΜ10 molecule was obtained due to effective quenching (second column on the left). After cleavage by при-9, the quenching of the fluorescence of the fragment containing the fluorophore ceases (strong fluorescence appears) and fluorescence appears in the lower part of the gel. As shown by the example of a gel, tumor homogenates are also cleaved 8ΌΜ-10 to obtain a product with strong fluorescence. This reaction is blocked by the ΜΜΡ inhibitor, indicating that cleavage is due to the ΜΜΡδ associated with the tumor.

In FIG. Figure 5 shows the biodistribution of three fluorescent compounds 6 hours after the introduction of (IV) into the tail vein 2.9 nmol of each compound. 8ΌΜ-6 is characterized by a 5-fold higher distribution in the tumor compared to 8ΌΜ-1 and 8ΌΜ-2. Molecules for selective delivery of 1 and 2 contain the same amount of glutamate and arginine residues, which leads to the formation of a neutral network core, while 8ΌΜ-6 contains a 3+ charge due to the presence of more positively charged arginines.

In FIG. Figure 6 shows the visualization of the RKET emission ratio to determine the presence of cancer in the mouse lymph nodes. This emission ratio was obtained using equation 2, where Exp1 = 0.7 s, Exp2 = 4.1 s and k = 20. The right panel shows an image in a format that indicates a strong contrast between the metastatic node (a very large lymph node indicated by the lower left dark arrow) and non-metastatic lymph nodes (other arrows). A higher ratio is shown as lighter elements (metastatic) compared to darker elements of non-metastatic lymph nodes.

In FIG. 7 shows the results of ex νίνο determination of mouse tissue activity. 8ΌΜ-23 was incubated with activated tumor tissue homogenates and normal tissue muscle homogenates of the thigh. The enzymatic activity of tissues led to the cleavage of 8ΌΜ-23 and to a large increase in RKET (labeled primary tumor). This increase was the result of 8ΌΜ cleavage. Normal muscle tissue did not cleave 8ΌΜ-23.

In FIG. Figure 8 shows the results of ex νίνο determination of human tissue activity using the RKET emission coefficient. 8ΌΜ-25 was incubated together with homogenate samples of normal human breast tissue and human breast cancer tissue (\ UO2808. ^ 02821, \ UE2815. ^ 02817, \ UE2824). It was found that the enzymatic activity and cleavage of 8ΌΜ-25 is significantly greater in the cancerous tissue of a human breast than in normal human breast tissue (data with errors).

In FIG. Figure 9 shows the values of the RKET emission coefficient obtained by conducting ex νίνο analysis of human tissue. 8ΌΜ-25 and 8ΌΜ-32 were incubated with a sample of normal healthy human breast tissue (^ 02823) and human breast cancer tissue (^ 02808, ^ 02815). It was found that the enzymatic activity and cleavage of 8ΌΜ is significantly greater in human breast cancer than in normal human breast tissue.

In FIG. Figure 10 shows the values of the RKET emission coefficient by the example of positive and negative lymph nodes in the model of the metastatic lymph node of the mouse, which was treated with 8ΌΜ-24. Nodes were considered either positive or negative based on an analysis of H&E staining by a pathologist.

In FIG. Figure 11 shows the CBS curve obtained by changing the threshold value used to determine either a positive or negative prognosis of the formation of metastases in relation to the emission coefficient using 8ΌΜ-24 in the metastatic lymph node model. These data show high sensitivity and specificity in the diagnosis of malignant and healthy lymph nodes.

- 3,030,795

Information confirming the possibility of carrying out the invention

Molecules for selective delivery allow targeted delivery of therapeutic agents and / or imaging agents to specific cells and / or specific tissues. In some embodiments, molecules for selective delivery include (a) a molecular transfer or retention sequence (part B), (b) at least one delivery fragment (part Ό) associated with part A, B or X, (c) linker X and (d) a macromolecular carrier and (e) an acidic amino acid sequence (part A) that is effective in inhibiting or preventing cellular uptake or tissue retention.

In some embodiments, the cleavage of linker X, which leads to the separation of part A from part B, is effective in absorbing or retaining part B and attached sagdo cells and tissue.

However, molecules for selective delivery can undergo rapid pharmacokinetic clearance with a short plasma half-life, wide distribution, and slow leaching from many non-target tissues with non-specific uptake. Thus, there is a need for a molecule for selective delivery with increased ίη νίνο circulation, accumulation in the target tissue relative to non-target tissues, modulated by selectivity. In the case of imaging agents, there is a need to increase the contrast in the target tissue compared to the background tissue.

Some definitions.

In this application, the terms used have the following meanings, unless otherwise indicated.

The term “target” molecule as used refers to any agent (e.g., peptide, protein, nucleic acid polymer, aptamer or small molecule) that binds (e.g., attaches) to a target of interest. The target of interest may be tissue, cell, cell structure (e.g., organelle), protein, peptide, polysaccharide or nucleic acid polymer. In some embodiments, the target molecule is any agent that associates (for example, binds to one or more cancer cells of a subject).

Used in this application, the terms polypeptide, peptide and protein are used interchangeably and refer to amino acid polymer residues. These terms refer to polymers of amino acids of natural origin, as well as polymers of amino acids in which one or more amino acid residues are amino acid residues of a non-natural origin (for example, an amino acid analog). These terms encompass chains of amino acids of any length, including full-length proteins (namely, antigens) in which amino acid residues are linked by covalent peptide bonds. Used in this application, the term peptide refers to amino acid residues of the polymer, the length of which includes from 2 to about 50 residues. In some embodiments, the peptide contains from about 2, 3, 4, 5, 7, 9, 10, or 11 residues to about 50, 45, 40, 45, 30, 25, 20, or 15 residues. When an amino acid sequence is provided in this application, it implies both b-, Ό- or beta-amino acid versions of the sequence, as well as retro-, inversion and retro-inversion isoforms. Peptides also include polymeric amino acids in which one or more amino acid residues are an artificial chemical analogue of the corresponding amino acids of natural origin, as well as polymeric amino acids of natural origin. In addition, this term refers to amino acids joined by peptide bonds or other modified bonds (for example, when the peptide bond is replaced by an a-ester, β-ester, thioamide, phosphonamide, carbamate, hydroxylate groups and the like (see, for example, 8p1o1a, (1983) Sket. Vusket. Ltto Aabk apb Rgoyu 7: 267-357), where the amide is substituted with a saturated amine (see, for example, 8kg1e5 e1 a1., US patent No. 4496542, which is incorporated herein by reference, and publication Kakepgopp e1 a1. (1990) pp. 969-970 ίη Proc. 11, ^'1' Ltepsap Rerbbe §utro8sht, E8SOM §s1epse Rikkk rk TKE №1keg1apb8, etc.).

The term amino acid refers to natural and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that act as natural amino acids. Natural amino acids are amino acids that are encoded by the genetic code, as well as those amino acids that were later modified, such as hydroxyproline, γ-carboxyglutamate and O-phosphoserine. The term amino acid analogs refers to compounds that have the same basic chemical structure as natural amino acids, that is, when a carbon is attached to hydrogen, to a carboxyl group, an amino group, and a K group, for example, homoserine, norleucine, methionine sulfoxide. Such analogs contain modified K groups (for example, norleucine) or modified peptide backbones, but retain the same basic chemical structure as natural amino acids. Amino acid mimetics are chemical compounds that have a structure different from the general chemical structure of an amino acid, but which act in a manner similar to natural amino acids. Amino acids can be Ό-amino acids or b-amino acids.

- 4,030,795

In this application, amino acids can be denoted by well-known three-letter symbols or one-letter codes, which are recommended by the SHRLS-SH VyusNetuya1 Ioteps1a commission (IgG Sotpikkyup. Nucleotides can also be denoted by single-letter codes.

The person skilled in the art is well aware that individual substitutions, deletions or additions to the amino acid sequence of a peptide, polypeptide or protein that alter, add or remove one amino acid or a small number of amino acids in the encoded sequence are a conservatively modified version, where the change leads to amino acid substitution chemically similar amino acids.

Tables showing conservative substitution are well known in the art. Such conservatively modified variants are complemented by polymorphic variants and do not exclude interspecific homologues and alleles of the invention.

Used in this application, the term label refers to a molecule that facilitates the visualization and / or detection of the target molecule described in this application. In some embodiments, the label is a fluorescent substance.

The expression specifically binds when it refers to the interaction between the target molecule described in this application and the target (for example, purified protein, cancer cells or cancer tissue, a tumor or metastatic damage, metastases or lymph node or metastatic lymph node), refers to the formation of a relationship with high affinity between the targeted molecule and the target. In addition, this term means that the target molecule has a low affinity for non-target molecules.

The terms selective binding selectivity and the like. refers to the agent’s preference for interacting with one molecule over another molecule. Preferably, the interactions between the target molecule described in this application and the target are both specific and selective. It should be noted that according to some variants, such an agent is obtained in order to specifically bind two different, but still similar targets without binding to other undesirable targets.

The terms individual, patient, or subject are used interchangeably. In this application, they mean any mammal (i.e., species of any group, members of families and genus in the taxonometric classification of animals: chordates: vertebrates: mammals). In some embodiments, the mammal is human. None of these terms is limited and does not need to be limited to a situation characterized by the observation (5 and 15 days) (for example, permanent or periodic) of a health worker (for example, a doctor, registered nurse, nurse, paramedic, nurse or hospice worker.

The terms administer, administration and the like, used in this application, refer to methods that can be used to ensure the delivery of agents or compositions to a desired site of biological action. These methods include, but are not limited to, parenteral injection (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular, intravascular, intrathecal, intravitreal injection, infusion, or local injection). The administration procedure, which is usually used to administer the agents, and the administration methods used in this application are described, for example, in the publication Soobtap apb Stap, Tne RNagtasoodyu1 Vak15 oG Tnegareibsk, st. Eb .; Regdatop; and KettdYup'k, Rnattaseibs1 Zaepsek (sigget ebyp), Musk RnKypd Co., Eak! op, Ra.

The term pharmaceutically acceptable, as used in this application, refers to a material that does not eliminate the biological activity or properties of the agents described in this application and is relatively non-toxic (that is, the advantages of the material significantly outweigh the toxicity of the material). In some cases, a pharmaceutically acceptable material may be administered to the individual and not cause significant undesirable biological effects or not cause an adverse effect when interacting with any component of the composition in which it is contained.

Used in this application, the term surgery refers to any method that can be used to study, manipulate, modify or obtain an effect in tissue by physical intervention. These methods include, but are not limited to, open surgery, endoscopic surgery, laparoscopy, minimally invasive surgery, robotic surgery, and any procedure that can affect cancer tissue, such as tumor resection, cancer ablation, cancer staging, cancer diagnosis, staging lymph nodes, detection of signal lymph nodes, or cancer treatment.

The term “controlled surgery” as used in this application refers to any surgical procedure when the surgeon uses a visualizing agent to control surgical intervention.

Used in this application, the term cancer refers to any disease, including uncontrolled growth or proliferation of cells in the human body. Cancer is also characterized by the ability of cells to migrate from the original focus and spread to distant sites (i.e., metastasize). Cancer types include sarcomas, carcinomas, lymphomas, leukemia, blastomas, or tumor cells of the germ cells. Cancer can develop in many tissues, including, but not limited to, the lung, chest, ovaries, colon, esophagus, rectum, bones, prostate, brain, pancreas, bladder, kidney, liver, blood cells, lymph nodes, and stomach.

Molecules for selective delivery.

The invention provides a molecule for selective delivery, having the formula I [Ό a- with a] - A- [cm-M] -X-B - [with _in -Όβ] formula I, where X is a peptide linker that is capable of cleavage using protease;

A is a peptide with a sequence containing 5 consecutive glutamate residues;

B is a peptide with a sequence containing 8 consecutive arginine residues;

with _A , c _in and with _M , each independently, is an amino acid residue;

M is a polyethylene glycol polymer (PEG);

Ό _α and I _in are a pair of acceptor and donor fluorescent particles between which fluorescence resonant energy transfer by the Förster mechanism is possible; and where _[M-M] attached at any position to X, _{[IA A} -c] attached to any amino acid in A and _{[C-O] n} attached to any amino acid in the B, M is the polymer polyethylene glycol (PEG ) having an average molecular weight of 500, 1000, 2000, 5000 or 10000 Da.

In one embodiment of the invention with _A , c _, and _M , each independently selected from β-cysteine, β-glutamate, lysine and para-4-acetyl-β-phenylalanine. c _in is the residue of сте-cysteine.

In one embodiment of the invention, _A represents a β-glutamate or lysine residue.

In one embodiment of the invention, _M is the residue of para-4-acetyl-L-phenylalanine.

In one embodiment of the invention, X is capable of cleavage using matrix metalloproteinase.

In one embodiment of the invention, X contains an amino acid sequence that is capable of cleavage using MMP2, MMP7, MMP9 or MMP14. X contains an amino acid sequence selected from

PbOAo, PbO-C (te) -AO, EPAUUEZ, Ε3ΡΑΥΥΤΑ, ΌΡΕ3ΡΕ, REZREZ, BFkKe and EG0n <(Ac).

In one embodiment of the invention, X comprises the amino acid sequence PY-OO-AO.

In one embodiment of the invention, X contains the amino acid sequence of PbO-C (sce) AO.

In one embodiment of the invention, X contains the amino acid sequence of ERHAEMES.

In one embodiment of the invention, Ό _α and E _in mean Su5 and Su7.

In one embodiment of the invention, Ό _α and E _in mean Cy5 and 1EOue750.

In one embodiment of the invention, Ό _α and E _in mean Cy5 and 1EEEE800.

In one embodiment of the invention, Ό _α and E _in mean Cy5 and 1CO.

In one embodiment of the invention, the molecule of formula (I) is

3ΌΜ-25.

Imaging agents.

In some embodiments, the imaging agent is a colorant. In some embodiments, the imaging agent is a fluorescent moiety. In some embodiments, the fluorescent moiety is selected from: a fluorescent peptide, a fluorescent dye, a fluorescent material, or a combination thereof.

All fluorescent groups are encompassed by the term fluorescent moiety. The specific examples of fluorescent fragments given in this application do not limit the fluorescent substances that can be used with the targeted molecules described in this application.

Examples of fluorescent dyes include, but are not limited to, xanthenes (for example, rhodamines, rhodols, and fluoresceins and their derivatives); bimans; coumarins and their derivatives (for example, umbelliferone and aminomethyl coumarins); aromatic amines (e.g., dansil, squared dyes); benzofurans; fluorescent cyanines; indocarbocyanins; carbazoles; dicyanomethylene pyranes; polymethine; oxabenzanthran; xanthene; pyrilia; carbostyl; perylene; acridone; quinacridone; rubren; anthracene; coronine; phenanthrene; pyrene; butadiene; stilbene; porphyrin; phthalocyanine; chelated

- 6,030,795 complexes of lanthanide metals; rare earth chelate complexes; as well as derivatives of such dyes.

Examples of fluorescent dyes include, but are not limited to, 5-carboxyfluorescein, fluorescein-5-isothiocyanate, fluorescein-6-isothiocyanate and 6-carboxyfluorescein.

Examples of rhodamine dyes include, but are not limited to, tetramethylrodamine-6 isothiocyanate, 5-carboxytetramethylrodamine, 5-carboxyiodole derivatives, tetramethyl- and tetraethylrodamine, diphenyldimethyl- and diphenyldiethylrodamine, dinaphthylrodamine and rhodamine 101 sulfonyl titanium chloride (commercially available Sulfonyl chloride).

Examples of cyanine dyes include, but are not limited to, Su3, SuZV, Su3.5, Su5, Su5.5, Su7, 1KBUE680, A1ekha P1iog 750, 1Kbuye800CA, 1CO.

Examples of fluorescent peptides include OPP (green fluorescent protein) or derivatives of CPP (e.g. EBRD, EBPP2, azurite, tKa1ata1, ECPP, azure, CyPe1, URP, citrine, Ueyik, URE1).

Fluorescent labels are detected in any suitable way. For example, a fluorescence tag can be detected by excitation of fluorochrome with the appropriate wavelength and detection of fluorescence, for example, under a microscope, by visual observation, using photographic film, using electronic detectors such as charge coupled devices (CSCs), photomultiplier tubes etc.

In some embodiments, a label is introduced into the imaging agent: a positron-active isotope (e.g., ¹⁸ P) for positron emission tomography (PET), a gamma-active isotope (e.g., ^99t Tc) for single-photon emission tomography (8REST), or a paramagnetic molecule or nanoparticle (for example, chelate Sb ³⁺ or a nanoparticle coated with magnetite) for magnetic resonance imaging (MK1).

In some embodiments, gadolinium chelate, iron oxide particles, supermagnetic iron oxide particles, ultrafine paramagnetic particles, manganese chelate, or gallium containing agent are introduced into the imaging agent as a label.

Examples of gadolinium chelates include, but are not limited to, diethylene triamine pentaacetic acid (BTPA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (BOTA), and 1,4,7-triazacyclononan-D, No. , No.'-triacetic acid (DOTA).

In some embodiments, the imaging agent is a near-infrared fluorophore, luciferase (firefly luciferase, bacterial luciferase or intestinal luciferase) or other luminescent molecule for bioluminescent imaging or vesicles filled with perfluorocarbon for the ultrasound method.

In some embodiments, the imaging agent is a nuclear probe. In some embodiments, the imaging agent is a radionuclide probe for 8REST or PET. In some embodiments, the imaging agent is selected from technetium chelate, copper chelate, radioactive fluorine, radioactive iodine, and indium chelate.

Examples of Tc chelates include, but are not limited to, ΗΥΝΙΟ BTRA and BOTA. In some embodiments, the imaging agent contains a radioactive moiety, for example, a radioactive isotope such as ²¹¹ A1, ¹³¹ Ι, ¹²⁵ Ι, ⁹⁰ Υ, ¹⁸⁶ Ke, ¹⁸⁸ Ke, ¹⁵³ 8t, ²¹² Βί, ³² P, ⁶⁴ Cu, radioactive isotopes Li and others isotopes.

In some embodiments, a molecule for the selective delivery of Formula Ι containing an imaging agent is used in surgery under imaging control. In some embodiments, the molecule for selective delivery is preferably localized to cancerous or other unwanted tissues (i.e., to necrotic tissues). In some embodiments, a molecule for selective delivery of Formula Ι containing an imaging agent is used in imaging surgery to remove a tumor in case of colorectal cancer. According to some options, during surgery under visual control, the use of molecules for selective delivery enables the surgeon to cut out as little as possible a section of healthy (i.e. non-cancerous) tissue. According to some options, in surgery under visual control, the use of molecules for selective delivery enables the surgeon to see and excise a larger portion of the carcinogenic tissue than he would be able to excise in the absence of molecules for selective delivery. According to some options, such surgery is performed under fluorescence control.

Macromolecular carriers.

The term “carrier” means an inert molecule that modulates the plasma half-life, solubility, or biodistribution. In some embodiments, the carrier modulates the plasma half-life of the molecule for the selective delivery described in this application. In some embodiments, the carrier modulates the solubility of the molecule for the selective delivery described herein. In some embodiments, the carrier modulates the biodistribution of the molecule for the selective delivery described herein.

In some embodiments, the carrier decreases the extent of absorption of the molecule for selective delivery by non-target cells or tissues. In some embodiments, the carrier

- 7,030,795 reduces the degree of absorption of the molecule for selective delivery of cartilage. In some embodiments, the carrier reduces the absorption of the molecule for selective delivery by joints associated with the target tissue.

In some embodiments, the carrier enhances the absorption of the molecule for selective delivery by target cells or tissues. In some embodiments, the carrier reduces the absorption of the molecule for selective delivery by the liver when the target tissue is located nearby. In some embodiments, the carrier reduces the absorption of the molecule for selective delivery by the kidneys. In some embodiments, the carrier promotes the uptake of the molecule for selective delivery of cancerous tissue. In some embodiments, the carrier promotes absorption of the molecule for selective delivery in the lymphatic channels and / or lymph nodes.

In some embodiments, the carrier increases the half-life in plasma by decreasing glomerular filtration.

In some embodiments, the carrier modulates the half-life in plasma by increasing or decreasing metabolism or degradation by a protease.

In some embodiments, the carrier enhances tumor uptake due to increased permeability and retention (EPK) of the tumor vasculature.

In some embodiments, the carrier increases the solubility of the molecules for selective delivery in water.

According to some variants, any Μ is independently connected directly or indirectly (for example, via _M ) with _A , B or X.

According to some variants, any Μ is independently connected to _A through the Ν-terminal polyglutamate.

According to some variants, any Μ is independently connected to the _A (or Ν-terminal polyglutamate) covalent bond.

In some embodiments, any Μ is independently connected to _B via the C-terminal polyarginine.

In some embodiments, any Μ is independently linked to a _B (or C-terminal polyarginine) covalent bond.

According to some variants, any Μ is independently connected directly or indirectly with linkers between X and A, X and B, B and CGO ends and A and CGO ends.

In some embodiments, the covalent bond is an ether bond, a thioether bond, an amine bond, an amide bond, an oxime bond, a carbon-carbon bond, a carbon-nitrogen bond, a carbon-oxygen bond, or a carbon-sulfur bond.

In some embodiments, Μ is selected from a protein, synthetic or natural polymer, as well as a dendrimer.

In some embodiments, Μ is selected from dextran, polyethylene glycol (PEC) (for example, PEC with a molecular weight of 5 kDa, PEC with a molecular weight of 12 kDa, PEC with a molecular weight of 20 kDa, PEC with a molecular weight of 30 kDa and PEC with a molecular weight of 40 kDa) Albumin or combinations thereof.

In some embodiments, Μ is RES.

In some embodiments, Μ has a molecular weight of between 50 and 70 kDa.

In some embodiments, the molecule for selective delivery is coupled to albumin.

In some embodiments, albumin is obtained from a glomerular filtrate under normal physiological conditions.

In some embodiments, the molecule for selective delivery contains a reactive group, such as a malimide group, which can form a covalent conjugate with albumin. An albumin-containing selective delivery molecule leads to an increased accumulation of cleaved molecules for selective delivery in tumors depending on the degree of cleavage.

In some embodiments, albumin conjugates have good pharmacokinetic properties.

In some embodiments, the molecule for selective delivery is coupled to PEC.

In some embodiments, the molecule for selective delivery is coupled to a RES with a molecular weight of 5 kDa. In some embodiments, the molecule for selective delivery is coupled to a RES with a molecular weight of 12 kDa.

In some embodiments, the molecule for selective delivery is coupled to a PEC with a molecular weight of 20 kDa.

In some embodiments, a PEC with a molecular weight of 30 kDa has a longer half-life compared to free peptides.

In some embodiments, the molecule for selective delivery is coupled to a RES with a molecular weight of 20-40 kDa, which is characterized by hepatic or renal clearance.

In some embodiments, the molecule for selective delivery is coupled to dextran.

In some embodiments, the molecule for selective delivery is coupled to dextran with a molecular weight of 70 kDa.

In some embodiments, the dextran conjugates are a mixture of oligomers with various

- 8,030,795 molecular weights and are difficult to synthesize and clean reproducibly.

In some embodiments, the molecule for selective delivery is coupled to streptavidin.

In some embodiments, the molecule for selective delivery is coupled to a fifth generation dendrimer RAMAM.

In some embodiments, the medium is blocked.

In some embodiments, blocking the carrier improves pharmacokinetics and reduces cytotoxicity of the carrier by adding hydrophilicity.

In some embodiments, the blocking agent is selected from: acetyl group, succinyl group, 3-hydroxypropionyl group, 2-sulfobenzoyl group, glycidyl group, RES-2, REO-4, RES-8, and RES-12.

Part X (linkers).

In some embodiments, a linker consisting of one or more amino acids is used to couple the peptide sequence A (namely, a sequence designed to inhibit the delivery action of peptide B) and peptide sequence B.

Typically, the peptide linker will not have a specific biological activity that is different from the activity of attaching a molecule or maintaining some minimum distance or other spatial relationship between them. However, the amino acids that make up the linker can be selected so that they affect some property of the molecule, including coagulation, net charge, or hydrophobicity.

In living cells, the intact molecule for selective delivery described in this application will be ineffective for cleavage of X in healthy cells, since part A. preventing penetration into the cell will be ineffectively cleaved by intracellular enzymes in healthy cells because it will not be absorbed and will not will gain access to such intracellular enzymes. The delivery molecule described in this application may be unable to enter the cell due to the presence of part A. However, when the cell is damaged or diseased (for example, carcinogenic cells, hypoxic cells, ischemic cells, apoptotic cells, necrotic cells), such intracellular enzymes leak out of the cell and A. will be cleaved. This allows the part in or the delivery group to get into the cell, increasing the targeted delivery of part B and / or the group for delivery of Ό to neighboring cells.

In some embodiments, X is cleaved in the extracellular space.

In some embodiments, the fact that capillaries often pass fluid around tumors and other injured sites, which increases the ability of high molecular weight molecules (i.e., molecules with a molecular weight of about 30 kDa or more) to enter the interstitial compartment.

In some embodiments, X. linker cells that do not express a relevant protease, but which are located directly next to the expressing cells, pick up delivery groups from the molecule for selective delivery, because linker X linkage usually occurs in the extracellular space.

In some embodiments, such nonspecific targeting is beneficial in treating tumors due to the heterogeneity of cell phenotypes and the desire to destroy as many suspicious cells as possible.

In some embodiments, X is a cleavable linker.

In some embodiments, X is flexible.

In some cases, X is hard.

In some embodiments, the linker has a linear structure.

In some embodiments, the linker has a non-linear structure.

In some embodiments, the linker has a branched structure.

In some embodiments, the linker has a cyclic structure.

In some embodiments, the length X is from about 5 to about 30 atoms.

In some embodiments, the length of X is about 6 atoms.

In some embodiments, the length of X is about 8 atoms.

In some embodiments, the length of X is about 10 atoms.

In some embodiments, the length of X is about 12 atoms.

In some embodiments, the length of X is about 14 atoms.

In some embodiments, the length of X is about 16 atoms.

In some embodiments, the length of X is about 18 atoms.

In some embodiments, the length of X is about 20 atoms.

In some embodiments, the length of X is about 25 atoms.

In some embodiments, the length of X is about 30 atoms.

In some embodiments, the linker connects the peptide portion A (i.e., the peptide sequence that prevents cellular uptake) to the peptide portion B (i.e., the delivery sequence) via a covalent bond.

- 9,030,795

In some embodiments, the covalent bond is an ester bond, a thioether bond, an amine bond, an amide bond, an oxime bond, a hydrazone bond, a carbon-carbon bond, a carbon-nitrogen bond, a carbon-oxygen bond, or a carbon-sulfur bond.

In some embodiments, X contains a peptide group. The peptide group consists of L-amino acids and / or Ό-amino acids.

In some embodiments, Ό-amino acids are preferred in order to minimize immunogenicity and non-specific cleavage of non-specific peptidases or proteases.

It is known that cellular uptake of oligo-O-arginine sequences is as effective as uptake of oligo-arginines or better.

In some embodiments, linker X is for cleavage under special conditions or in a particular environment. In preferred embodiments, linker X is cleaved under physiological conditions. The cleavage of such linker X can be accelerated by the use of specific pathological signals or a specific medium relative to the cells into which delivery groups are desired. Obtaining linker X for cleavage under specific conditions, for example, using a specific enzyme, allows targeted cell uptake at a specific location where such conditions are obtained. Thus, one of the key methods for specifically targeting cell uptake with molecules for selective delivery of the desired cells, tissues or regions is to obtain the linker portion X, which is cleaved under the conditions available near such target cells, tissues or regions.

In some embodiments, X is a pH sensitive linker.

In some embodiments, X is cleaved at basic pH values.

In some embodiments, X is cleaved at acidic pH.

In some embodiments, X is cleaved by a protease, matrix metalloproteinase, or a combination thereof.

In some embodiments, X is cleaved by a reducing agent.

In some embodiments, X is split by MMP. The hydrolytic activity of matrix metalloproteinases (MMPs) is organically associated with the invasive migration of metastatic tumor cells. In some cases, MMPs are found near inflammation sites. In some cases, MMPs are found near the site of an apoplexy stroke (that is, a disorder characterized by brain damage, accompanied by a decrease in blood flow).

Thus, the uptake of molecules possessing the features of the invention is capable of directing the uptake of groups for delivery (at least one fragment фраг) by specific cells, tissues or regions containing active MMPs in the extracellular environment. In some embodiments, linker X includes the amino acid sequences PbO-C (Me) LO (§ EC GO N0: 1), REOLO (§ EC GO N0: 2), which are cleaved by the enzymes metalloproteinases MMP-2, MMP-9 or MMP-7 ( MMP8 involved in the development of cancer and inflammation).

In some embodiments, linker X is cleaved by proteolytic enzymes or in the presence of a reducing agent in the environment, which can be detected near cancer cells. Such an environment or such enzymes are usually not found near normal cells.

In some embodiments, X is cleaved by serine proteases, including, but not limited to, thrombin.

In some embodiments, X is cleaved in or near tissues suffering from hypoxia. In some embodiments, cleavage in or near hypoxic tissues facilitates targeting of cancer cells and cancer tissues, heart attack sites, and other hypoxic sites.

In some embodiments, X contains a disulfide bond.

In some embodiments, the linker X containing the disulfide bond is preferably cleaved at the hypoxic sites and thus targets the delivery of groups for delivery to cells at that site. It is believed that hypoxia causes greater resistance of cancer cells to radiation and chemotherapy, and also initiates angiogenesis. In a hypoxic environment in the presence of, for example, fluid-permeable cells or necrotic cells, free thiols and other reducing agents become available outside the cells, while O2 typically supports oxidation in the extracellular environment and, by definition, decomposes.

In some embodiments, this shift in the redox balance accelerates the reduction and cleavage of the disulfide bond in linker X. In addition to the disulfide bonds, which are advantageous at thiol-disulfide equilibrium, in linker X, designed so that it cleaves in a hypoxic environment , groups including quinone groups that decompose upon reduction to hydroquinones are used.

In some embodiments, linker X is cleaved in a necrotic environment. Necrosis often leads to the release of enzymes or other cell components that can be used to trigger cleavage of the X linker.

- 10,030,795

In some embodiments, cleavage of linker X by necrotic enzymes (e.g., calpains) allows groups for delivery to be absorbed by diseased cells and neighboring cells that have not yet completely passed fluid.

In some embodiments, linker X is labile in an acidic environment.

In some embodiments, linker X contains an acetal group or a vinyl ester group. Acidosis is observed in areas of damaged or hypoxic tissue due to Warburg's shift from oxidative phosphorylation to anaerobic glycolysis and the formation of lactic acid.

In some embodiments, acidosis is used to trigger uptake of groups for delivery by replacing in some arginine residues with histidine residues that become cationic only at pH values less than 7.

It should be borne in mind that the linker described in this application may include non-standard amino acids, such as, for example, hydroxylisine, desmosin, isodesmosin or other non-standard amino acids. The linker described in this application may contain modified amino acids, including post-translationally modified amino acids, for example methylated amino acids (e.g. methylhistidine, methylated forms of lysine, etc.), acetylated amino acids, amidated amino acids, formulated amino acids, hydroxylated amino acids, phosphorylated amino acids or other modified amino acids. The linker described in this application may also include fragments of peptide mimetics, including parts linked by non-peptide groups, and amino acids linked by non-amino acid parts or attached to them.

In some embodiments, linker X comprises an amino acid sequence selected from

REOBAO (8ΕΟΙΡΝΟ: 2), REO-C (she) -AO (8ΕΟΙΡΝΟ:

1), CREAEAKZ (8EO ΙΡ N0: 7), Ε8ΡΑΥΥΤΑ (ZEO GO N0: 8), ΌΡΚδΡΕ (ZEO GO N0: 9),

RRKZR (8Е0 ГО N0: 10), ВЕРЬКЕ (8Е0 ГО N0: 11) and ВЕЕЕК (Ас) (8Е0 ГО N0: 12).

In some embodiments, linker X contains the amino acid sequence POPAO (8E0 GO N0: 2).

In some embodiments, linker X contains the amino acid sequence POC (te) -AC (8E0 GO N0: 1).

In some embodiments, linker X contains the amino acid sequence RB-CACS (8ES GO N0: 27), where x is the residue of any amino acid (natural or non-natural).

In some embodiments, linker X contains the amino acid sequence KLABAK8 (8E0 GO N0: 7).

In some embodiments, linker X contains the amino acid sequence E8PAAΥΤΑ (8ES GO N0: 8).

In some embodiments, linker X contains the amino acid sequence ΌΡΚ8ΡΕ (8E0 GO N0: 9).

In some embodiments, linker X contains the amino acid sequence PPK8RB (8E0 GO N0: 10).

In some embodiments, linker X contains the amino acid sequence of the UNCCD (8E0 ΙΌ N0: 11).

In some embodiments, linker X contains the amino acid sequence KEOEK (Ac) (8ES GO N0: 12).

In some embodiments, linker X contains a peptide selected from PK (8 / T) (B / 1) (8 / T) (where the letters in parentheses indicate that any one of these amino acids may be in this position in the sequence)

ΟΟΑΑΝΕνΚθθ (ZEO GO N0: 28); 3ΟΚΙΟΡΕΚΤΑ (8Е0 GO N0: 29); ZOKZA (8Ε0ΙΡΝ0: 30); ORIO (8E0

GO N0: 31); ARAB (8E0 GO N0: 32); RK; P1C (E1) PP (8E0 GO N0: 33), where C (E1) is 8-ethylcysteine (a cysteine residue with an ethyl group attached to a thiol group) and - indicates a typical cleavage site in this and subsequent sequences);

OORVSBRO (8E0 GO N0: 34); N88KBS (8E0 Π) ΝΟ: 35); BUBA-ZZZRSRG (8E0 GO N0:

36); 0ν8 (} ΝΥ-Ρΐν0 (8Е0 ГО N0: 37); СУУОА-ЗСЕЕА (8Е0 ГО N0: 38); ί (Ρίρ) Κ-8, where ί is the residue of Ό-phenylalanine and Ρϊρ is the remainder of piperidinocarboxylic acid (pipecolic acid, proline analogue containing a six-membered ring);

□ EUO (8Е0 GO N0: 39). SAENOS (8E0 GO N0: 40); CREAEAK8 (8E0 GO N0: 7) or a combination thereof.

In some embodiments, X is cleaved under hypoxic conditions.

In some embodiments, X contains a disulfide bond.

- 11 030795

In some embodiments, X contains a quinine residue.

According to some variants, X is cleaved off under necrotic conditions.

In some embodiments, X contains a molecule that is cleaved by calpain.

In some embodiments, X contains 6-aminohexanoyl, 5- (amino) -3-oxapetanoyl, or a combination thereof.

In some embodiments, X contains a disulfide bond.

In some embodiments, the linker is alkyl.

In some embodiments, the linker is heteroalkyl.

In some embodiments, the linker is alkyl.

In some embodiments, the linker is alkylene.

In some embodiments, the linker is alkenylene.

In some embodiments, the linker is alkynylene.

In some embodiments, the linker is heteroalkylene.

The term alkyl refers to an aliphatic hydrocarbon group. Alkyl may be saturated or unsaturated alkyl. Depending on the structure, the alkyl group may be a monoradical or a diradical (i.e., an alkylene group).

Alkyl may contain from 1 to 10 carbon atoms (wherever it is indicated, a numerical range, such as from 1 to 10, refers to each number in this interval, for example, a range from 1 to 10 carbon atoms means that the alkyl group may contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., and may contain 10 carbon atoms, although this definition also covers the case when the number of carbon atoms is not indicated in the coercion of the term alkyl).

The alkyl group may also be lower alkyl containing from 1 to 6 carbon atoms. The alkyl group in the compounds described in this application may be designated as C ₁ -C ₄ alkyl or the like. For example, the designation C ₁ -C ₄ alkyl indicates that the alkyl chain contains one to four carbon atoms, i.e. the alkyl group is selected from methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, ethenyl, propenyl, butenyl, and the like.

In some embodiments, the linker comprises a ring structure (e.g., aryl). Used in this application, the term ring refers to any covalent cyclic structure. Rings include, for example, carbocycles (e.g., aryls and cycloalkyls), heterocycles (e.g., heteroaryls and non-aromatic heterocycles), aromatic groups (e.g., aryls and heteroaryls), and non-aromatic rings (e.g., cycloalkyls and non-aromatic heterocycles). Rings may be substituted. Rings may be monocyclic or polycyclic.

As used in this application, the term aryl refers to an aromatic ring in which each of the atoms forming the ring is a carbon atom. Aryl rings may be formed by five, six, seven, eight, nine or more than nine carbon atoms. Aryl groups may be substituted. Examples of aryl groups include, but are not limited to, phenyl, naphthalenyl, phenanthrenyl, anthracenyl, fluorenyl, and indenyl. Depending on the structure, the aryl group may be a monoradical or a diradical (i.e., an arylene group).

The term cycloalkyl refers to a monocyclic or polycyclic non-aromatic radical in which each of the atoms forming a ring (for example, skeletal atoms) is a carbon atom. Cycloalkyls may be saturated or partially unsaturated. Cycloalkyl groups include groups containing from 3 to 10 atoms in the ring. Cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.

In some embodiments, the ring is cycloalkane.

In some embodiments, the ring is cycloalkene.

In some embodiments, the ring is aromatic. The term aromatic refers to a planar ring containing a delocalized π-electron system containing 4η + 2π electrons, where η is an integer. Aromatic rings can be formed by five, six, seven, eight, nine or more than nine atoms. Aromatic rings may be substituted. The term aromatic encompasses both carbocyclic aryl (e.g., phenyl) and heterocyclic aryl (or heteroaryl or heteroaromatic) groups (e.g., a pyridine group). This term includes a monocyclic or fused polycyclic ring (i.e., rings that have common adjacent pairs of carbon atoms) groups.

In some embodiments, the ring is a heterocycle. The term heterocycle refers to heteroaromatic and heteroalicyclic groups containing from one to four heteroatoms, each of which is selected from O, δ and Ν, and each heterocyclic group contains from 4 to 10 atoms in the ring system, provided that the ring in this group does not contain two adjacent atoms of O or δ. Non-aromatic heterocyclic groups include groups containing only 3 atoms in the ring system, but aromatic heterocyclic groups must contain at least 5 atoms in their ring system. Heterocyclic groups include benzofused

- 12,030,795 ring systems. An example of a 3 membered heterocyclic group is aziridinyl. An example of a 4 membered heterocyclic group is azetidinyl (derived from azetidine). An example of a 5 membered heterocyclic group is thiazolyl. An example of a 5 membered heterocyclic group is pyridyl, and an example of a 10 membered heterocyclic group is quinolinyl. Examples of non-aromatic heterocyclic groups include pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidinyl, morpholinyl, thiomorpholinyl, thiooxanyl, piperazinyl oxanediene azinide, , 3,6-tetrahydropyridinyl, 2pyrrolinyl, 3-pyrroinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofenyl uranyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, 3-azabicyclo [3.1.0] hexanyl, 3-azabicyclo [4.1.0] heptanyl, 3H-indolyl and quinolisinyl. Examples of aromatic heterocyclic groups include pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl azinolinyl, indinyl, indinyl, indinol, indole, indole, indole, indole, indole, indole, indole, indole, indole, indole, indole, indole, indole, indole, indole, indole, indole, indole, indole, indole, indole, indole, indole, indole, indole, indole, indole, indole, indole, indole, indole, indole, indole, indole, indole, indole, indole, indole, indole, indole, indole, indole, indole, indole, indole, indole, indole, indole, indole, indole, indole, indole, indoline , pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, furazanil, benzofurazanil, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl and furoopyridinyl. The above groups can be C-linked or Ν-linked, where possible. For example, a pyrrole-based group may be pyrrol-1-yl (Ν-attached or pyrrol-3-yl (Co-attached). Further, the imidazole-based group may be imidazol-1-yl or imidazol-3-yl ( both Ν-attached) or imidazol-2-yl, imidazol-4-yl or imidazol-5-yl (all Co-attached) Heterocyclic groups include benzofused ring systems and ring systems substituted with one or two oxo groups (= 0), such as pyrrolidin-2-one. Depending on the structure, a heterocyclic group may be be a monoradical or a diradical (i.e. a heterocycle group).

In some embodiments, the ring is fused. The term fused refers to structures in which two or more rings have one or more common bonds.

In some embodiments, the ring is a dimer.

In some embodiments, the ring is a trimer.

In some embodiments, the ring is substituted.

The term carbocyclic or carbocycle refers to a ring, where each of the atoms forming the ring is a carbon atom. Carbocycles include aryl and cycloalkyl. This term distinguishes a carbocycle from a heterocycle (heterocyclic ring) in which the ring contains at least one atom other than a carbon atom (i.e., is a heteroatom). Carbocycles include aryl and cycloalkyl. This term distinguishes a carbocycle from a heterocycle (heterocyclic ring) in which the ring contains at least one atom other than a carbon atom (i.e. a heteroatom). Heterocycles include heteroaryl and heterocycloalkyl. Carbocycles and heterocycles may be substituted.

In some embodiments, the linker is substituted. The term a substituted or an optionally substituted group means that the group may be substituted with one or more additional group (s), and each independently selected from C1-C ₆ alkyl, C ₃ -C ₈ cycloalkyl, aryl, heteroaryl, C2-S6geteroalitsiklicheskoy group , hydroxyl, C1-C6 alkoxy, aryloxy, C1-S6alkiltio, arylthio, _C1-C6 alkilsulfoksidnoy group arilsulfoksidnoy group, a _C1-C6 alkylsulfonyl group, arylsulfonyl group, cyano, halogen, C ₂ -C ₈ acyl, C ₂ -C ₈ acyloxy, nitro, _C1-C6 haloalkyl, O S6ftoralkila and amino, including C1-S6al ylamino and protected derivatives thereof. For example, possible substituents may be L3K3, where each L3 is independently selected from the bond, -O-, -C (= 0) -, -8-, -8 (= 0) -, -8 (= 0) 2-, - ΝΗ-, -ΝΗ0 (= 0) -, -0 (= 0) ΝΗ-, 5 (= 0) 2ΝΗ-, -ΝΗ5 (= 0) 2-, -00 (= 0) ΝΗ-, -ΝΗϋ (= 0) 0-, - (C ₁ -C ₆ alkyl) - or - (C ₂ -C ₆ alkenyl) -; and each K3 is independently selected from H, (C1C4 alkyl), (C3-C8 cycloalkyl), heteroaryl, aryl, and C1-C6 heteroalkyl. Possibly substituted non-aromatic groups may, as substituents, contain one or more oxo groups (= 0). The protecting groups that are used to prepare the protected derivatives of the above substituents are well known to those skilled in the art.

In some embodiments, the molecules for selective delivery described herein contain one linker. The use of a single mechanism for mediating the uptake of both imaging and therapeutic delivery agents is especially important since imaging with harmless trace amounts can be used for testing if the further therapeutic dose is to be properly concentrated in the target tissue.

In some embodiments, the molecules for selective delivery described herein contain multiple linkers. When the molecule for selective delivery described in this application includes many linker groups, the separation of part A from the remaining parts of the molecule requires the cleavage of all linker groups X. The cleavage of the set of linkers X can be simultaneous or sequential. The set of linkers X may include groups X having different specificities, so that the separation of part A from the rest of the molecule requires more than one condition or more than one medium (extracellular signals) for this molecule. Cleavage of the plurality of linkers X thus serves as a detector of combinations of such extracellular signals. For example, a molecule for selective delivery may include two parts of the linkers Xa and Xb connecting the main part B to the acidic part A. Both linkers Xa and Xb must be cleaved before the acidic part A is separated from the main part B and the delivery group C (if any ) to enter the cage. It should be borne in mind that the linker site can connect either to the main part B or to the delivery group C, regardless of the presence of another linker, and that, when desired, more than two linkers X can be contained.

Combinations of two or more X linkers can be used to further modulate the targeting and delivery of molecules to desired cells, tissues, or sites. Combinations of extracellular signals are used to expand or narrow the specificity of the cleavage of linkers X, when desired. When multiple linkers are connected in parallel, the specificity of cleavage is narrowed, since each linker must be cleaved before part A can separate from the rest of the molecule. When a plurality of linkers is connected in series, the cleavage specificity expands, since the cleavage of any one linker X allows part A to separate from the rest of the molecule. For example, in order to determine the presence of hypoxia or protease (i.e., the cleavage of X in the presence of a protease or the presence of hypoxia), the linker X must be such that the protease-sensitive or reduction-sensitive sites are placed in tandem so that the cleavage of each of them is sufficient to separate acidic parts A.

Or, in order to detect the presence of both proteases and hypoxia (that is, to effect the cleavage of linker X in the presence of both protease and hypoxia, but not in the presence of only one of these factors), linker X must be such as to place a proteasensitive site between at least one pair of cysteines that are linked by a disulfide bond to each other. In this case, both protease cleavage and disulfide bond reduction are required in order for the separation of part A.

Examples of molecules for selective delivery

In some embodiments, the invention provides molecules for the selective delivery of 8ΌΜ-14.

In some embodiments, the invention provides molecules for the selective delivery of 8ΌΜ-15.

In some embodiments, the invention provides molecules for the selective delivery of 8ΌΜ-23.

In some embodiments, the invention provides molecules for the selective delivery of 8ΌΜ-24.

In some embodiments, the invention provides molecules for the selective delivery of 8ΌΜ-25.

In some embodiments, the invention provides molecules for the selective delivery of 8ΌΜ-26.

In some embodiments, the invention provides molecules for the selective delivery of 8ΌΜ-27.

In some embodiments, the invention provides molecules for the selective delivery of 8ΌΜ-32.

In some embodiments, the invention provides molecules for the selective delivery of 8ΌΜ-35.

In some embodiments, the invention provides peptides P-3.

In some embodiments, the invention provides molecules for the selective delivery of 8ΌΜ-14.

In some embodiments, the invention provides molecules for the selective delivery of 8ΌΜ-15.

In some embodiments, the invention provides molecules for the selective delivery of 8ΌΜ-23.

In some embodiments, the invention provides molecules for the selective delivery of 8ΌΜ-24.

In some embodiments, the invention provides molecules for the selective delivery of 8ΌΜ-25.

In some embodiments, the invention provides molecules for the selective delivery of 8ΌΜ-26.

In some embodiments, the invention provides molecules for the selective delivery of 8ΌΜ-27.

In some embodiments, the invention provides molecules for the selective delivery of 8ΌΜ-32.

- 14,030,795

In some embodiments, the invention provides molecules for the selective delivery of 8ΌΜ-35.

In some embodiments, the molecule for selective delivery has a structure selected from δϋΜ-1, δϋΜ-2, δϋΜ-3, δϋλΙ-4. δϋΜ-5, δϋΜ-6, δϋΜ-7, δϋΜ-8, δϋΜ-9, δϋΜ-10, δϋΜ-11, δϋΜ-12, δϋΜ-13, δϋΜ-14, δϋΜ-15, δϋΜ-16, δϋΜ17, δϋΜ-18, δϋΜ-19, δϋΜ-20, δϋΜ-21, δϋΜ-22, δϋΜ-23, δϋΜ-24, δϋΜ-25, δϋΜ-26, δΏΜ-27, δϋΜ-28, δΏΜ-29, δϋΜ- 30, δΏΜ-31, δϋΜ-32, δΏΜ-33, δϋΜ-34, δΏΜ-35, δϋΜ-36, δϋΜ-37, δϋΜ-38, δϋΜ-39 and δϋΜ-40.

- 15,030,795

- 16,030,795

- 18,030,795

l

<l

ABOUT

L o

about

L

- 19 030795

ZO ₃ N

- 20,030,795

5О ₃ Н

- 21,030,795

- 22,030,795

- 23,030,795

- 24,030,795

5О ₃ Н

- 26,030,795 about

ω

ιζ? ° ο

$

V

ΖΙ

uh .....

hK ° η ζτ ° ί τζ

- 27,030,795

Additional modifications.

In some embodiments, the targeted molecules of the invention may be coupled to high molecular weight molecules that increase the multivalence and avidity of labeling. In some embodiments, the high molecular weight molecules are water soluble polymers. Examples of water soluble polymers include, but are not limited to, peptides, saccharides, vinyl polymers, polyethers, polyamines, polycarboxylic acids, and the like. In some embodiments, the water-soluble polymer is dextran, polyethylene glycol (PEC), polyoxyalkylene, polysialic acid. Starch or hydroxyethylated starch. Any suitable method is used to couple the peptides with water soluble polymers.

- 28 030795 (see Negtapzop O., Vyusoprda Tesbtziyes 2 ^pb Eb, Lsabetk Przegz, 1 pps. 2008).

Application methods

Molecules for the selective delivery of Formula I allow targeted delivery of therapeutic agents to specific cells and / or tissues. These molecules contain the main peptide sequence (B), which is intended for transfer through the cell membrane, an acid peptide sequence (A), which inhibits the absorption of peptide B by cells, linker X, which is cleaved under specific conditions, of the delivery group (at least O. and O _B ) associated with peptides A and B, or X as a macromolecular carrier. In some embodiments, cleavage of linker X results in the separation of peptide B from peptide A and the transfer of peptide B (and any delivery group attached thereto) across the cell membrane. In some embodiments, the molecules for the selective delivery of Formula I allow targeted delivery of one or more delivery groups (e.g., therapeutic agents or imaging agents) to cell tissue.

In some embodiments, a selective delivery molecule of formula I is provided [Ό a-c a] - A- [cm-M] -X-B - [st - Ov] formula I, where X is a peptide linker that is capable of cleavage by proteases;

A is a peptide with a sequence containing 5 consecutive glutamate residues;

B is a peptide with a sequence containing 8 consecutive arginine residues;

with _A , _B, and _M , each independently, is an amino acid residue;

M is a polyethylene glycol polymer (PEG);

□ _A and O _B are a pair of acceptor and donor fluorescent particles between which fluorescence resonant energy transfer is possible by the Förster mechanism; and where [with _M -M] is attached in any position to X, [O _A -c _L | attached to any amino acid in A and [with _B -O _B | attached to any amino acid in B, M is a polymer polyethylene glycol (PEG) having an average molecular weight of 500, 1000, 2000, 5000 or 10000 Da.

In one embodiment of the invention with _A , _B and _{C M} , each independently selected from O-cysteine, Oglutamate, lysine and para-4-acetyl-G-phenylalanine. c _B is an O-cysteine residue.

In one embodiment of the invention, _A represents a residue of O-glutamate or lysine.

In one embodiment of the invention, _M is the residue of para-4-acetyl-G-phenylalanine.

In one embodiment of the invention, X is capable of cleavage using matrix metalloproteinase.

In one embodiment of the invention, X contains an amino acid sequence that is capable of cleavage using MMP2, MMP7, MMP9 or MMP14. X contains an amino acid sequence selected from

PbOAO, PbO-C (she) -AO,

KRAYUKZ, Ε8ΡΑΥΥΤΑ, ORKZR, RKZR, WHO and KOEOK (Ac).

In one embodiment of the invention, X comprises the amino acid sequence of PPC.AC.

In one embodiment of the invention, X comprises the amino acid sequence of PPC-C (te) AC.

In one embodiment of the invention, X contains the amino acid sequence of the HRCLL \\ 'K8.

In one embodiment of the invention, O _A and O _{B are} Cy5 and Cy7.

In one embodiment of the invention, O _A and O _B mean Cy5 and PCOue750.

In one embodiment of the invention, O _A and O _B mean Cy5 and PK0ue800.

In one embodiment of the invention, O _A and O _{B are} Cy5 and 1CC.

In one embodiment of the invention, the molecule of formula (I) is

δϋΜ-25.

Target tissue.

In some embodiments, the target tissues of interest are cancerous tissues.

- 29,030,795

In one embodiment of the invention, the tissue is breast cancer tissue, colorectal cancer tissue, squamous cell carcinoma tissue, prostate cancer tissue, melanoma tissue, or thyroid cancer tissue.

In one embodiment of the invention, the tissue is breast cancer tissue.

In one embodiment of the invention, the tissue is colorectal cancer tissue. In some embodiments, the cancer (cancer) is AIDS-associated cancer (e.g., AIDS-associated lymphoma), anal cancer, basal cell carcinoma, bile duct cancer (e.g., extrahepatic bile duct cancer), bladder cancer, bone cancer (osteosarcoma and malignant fibrous histiocytoxanthoma), breast cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer (e.g. uterine cancer), ependymoma, esophageal cancer, eye cancer (e.g. intraocular melanoma and retinoblastoma), stomach cancer, a germ cell tumor (for example, extracranial cancer, extragenosal cancer, ovarian cancer), head and neck cancer, leukemia, lip and oral cancer, liver cancer, lung cancer (for example, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and squamous cell lung cancer), ovarian cancer, pancreatic cancer, pituitary tumor, prostate cancer, liver cancer, skin cancer, small intestine cancer, squamous cell cancer, testicular cancer, throat cancer, thyroid cancer, urethral cancer ( urethra ala) and post-transplant lymphoproliferative syndrome (PTL).

In some embodiments, the cancer is a lymphoid cancer (e.g., lymphoma).

In some embodiments, the cancer is a B-cell cancer.

In some embodiments, the cancers include a precursor of extracellular cancers (e.g., a precursor of B-cell / cell lymphoblastic / lymphoblastic leukemia / lymphoma) and peripheral B-cell cancers (e.g., B-cell chronic lymphocytic leukemia / pro-lymphocytic leukemia / small cell lymphocytic lymphoma) (small cell lymphocytic (8b) non-Hodgkin's lymphoma (LUN, NHL), lymphoplasmacytoid lymphoma / immunocytoma, mantle cell lymphoma, lymphoma foul lymphoma lymphoma (for example, according to the cytological classification: I (small cell), II (mixed small and large cell), III (large cell), and / or subtypes: diffuse and predominantly small cell type), low degree of malignancy (underdeveloped) / follicular non-Hodgkin lymphoma (ХНЬ), intermediate degree of malignancy / follicular ХНЬ, extracellular lymphoma of the marginal zone (for example, extra-nodal MAB type +/- monocytoid B cells) and / or nodular (for example +/- monocytoid B cells)), lymphoma of the spleen marginal zone and (e.g., +/- hairy lymphocytes), hairy cell leukemia, plasmacytoma / plasmacellular myeloma (e.g., myeloma and multiple myeloma), diffuse large-cell B-cell lymphoma (e.g., primary mediastinal (thymic) B-cell lymphoma), diffuse XNI intermediate malignancy, Burkitt’s lymphoma, B-cell lymphoma of high malignancy, immunoblastic XBH type of high malignancy, lymphoblastic XHI of high malignancy, XHI from small cells with no split nuclei of a high degree of malignancy, massive HNB, AIDS-associated lymphoma and Waldenstrom macroglobulinemia).

In some embodiments, the cancer is T cell and / or putative ΝΚ cell cancer.

In some embodiments, the cancer is a precursor of T-cell cancer (a precursor of T-lymphoblastic / lymphoblastic lymphoma / leukemia) or the cancers are peripheral T-cell and ΝΚ-cell cancers (e.g., chronic lymphocytic leukemia / proliferative leukemia and leukemia from large granular leukocytes (LBC) (e.g., T-cell type and / or ΝΚ-cell type), cutaneous T-cell lymphoma (e.g., fungoid granuloma / Cesari syndrome), primary nonspecific T-cell lymphomas (e.g., by cytological classification (e.g., medium-cell, mixed medium- and large-cell tumors), large-cell, from lymphoepithelioid cells, a subtype of hepatosplenic γδ T-cell lymphoma, and subcutaneous panniculitic T-cell lymphoma), angioimmunoblastic T- cell lymphoma (AITL, AGBO), angiocentric lymphoma, intestinal T-cell lymphoma (for example, associated with +/- enteropathy), T-cell / Tcellular lymphoma / adult leukemia (ATL), anaplastic large-cell lim foma (ABC) (for example, from cells of the type ί'.Ό30 +. T-null), anaplastic large cell lymphoma and Hodgkin’s lymphoma).

In some embodiments, the cancer is Hodgkin's disease.

In some embodiments, the cancer is leukemia.

In some embodiments, the cancer is chronic myelocytic I (granulocytic) leukemia, chronic myelogenous and chronic lymphocytic

- 30,030,795 leukemia (CLL, Cb), acute lymphoblastic leukemia (lymphocytic leukemia, ALL, AB), acute myeloid leukemia, acute lymphocytic leukemia, and acute myelocytic leukemia (e.g., myeloblastic, promyelocytic, myelomonocytic monolithocyte leukemia).

In some embodiments, the cancer is a non-solid tumor or plasmacytoma.

In some embodiments, the cancer is extramedullary plasmacytoma, solitary myeloma, and multiple myeloma.

In some embodiments, plasmacytoma is multiple myeloma.

In some embodiments, the cancer is lung cancer.

In some embodiments, the cancer is prostate (prostate) cancer.

In some embodiments, prostate cancer is adenocarcinoma.

In some embodiments, prostate cancer is a sarcoma, a neuroendocrine tumor, small cell cancer, ductal cancer, or lymphoma.

In some embodiments, prostate cancer is stage A prostate cancer (cancer may not be palpable during rectal examination).

According to some embodiments of the invention, prostate cancer is stage B of prostate cancer (i.e., a tumor in the prostate gland contains more cells (tissue), which can be detected by rectal examination or by biopsy, which is performed due to the high PSA level (P8A)).

According to some embodiments of the invention, prostate cancer is stage C of prostate cancer (i.e., the cancer has spread beyond the prostate gland to adjacent tissues).

In some embodiments, prostate cancer is stage Ό prostate cancer. In some embodiments, prostate cancer is an androgen-independent prostate cancer (A1RS).

In some embodiments, prostate cancer is androgen-dependent prostate cancer.

In some embodiments, prostate cancer is immune to hormone therapy.

In some embodiments, prostate cancer is virtually immune to hormone therapy.

In some embodiments of the invention, prostate cancer is immune to chemotherapy.

In some embodiments, prostate cancer is metastatic prostate cancer.

According to some variants of the invention, the subject is a person in the body of which there is a gene, genetic mutation or polymorphism associated with prostate cancer (for example, РКА8Е / НРС1, ЕБАС2 / НРС2, 8К-А / М8К1, СНЕК2, ВКСА2, ΡΟΝ1, ΘΟΟ1, М1С- 1, TBK4 and ΡΤΕΝ), or there is one or more additional copies of the gene associated with prostate cancer.

In some embodiments, prostate cancer is HEK2-positive.

In some embodiments, prostate cancer is HEK2-negative.

In some embodiments, prostate cancer is metastasized and is characterized by circulating tumor cells.

Application for visualization.

Selective delivery of molecules of Formula I provides targeted delivery of imaging agents to specific cells and / or tissues (e.g., cancerous tissues). The molecules contain the main peptide sequence (B), which is designed so that it can be transported across the cell membrane or retained in tissue, an acidic peptide sequence (A) that inhibits the capture and retention of peptide B in cells, linker X, which cleaves in certain conditions visualizing agents associated with peptides A and B, or X and a high molecular weight carrier.

In some embodiments of the invention, cleavage of linker X allows peptide B to be separated from peptide A and makes it possible to transfer peptide B (and any associated imaging agents) across the cell membrane or to retain peptide B in tissue.

In some embodiments of the invention, selective delivery of the molecules of Formula I provides targeted delivery of one or more imaging agents to a cell or tissue.

According to some embodiments of the invention, selective delivery of an imaging agent to a cell or tissue allows a medical professional to visualize / obtain an image of a particular tissue.

In some embodiments of the present invention, methods are disclosed for delivering imaging agents to a target tissue, comprising contacting the target tissue with a molecule of formula I

- 31 030795 [β l-s a] - A- [cm-M] -X-B - [CB Ό β] formula I, where X is a peptide linker that is capable of being cleaved by a protease;

A is a peptide with a sequence containing 5 consecutive glutamate residues;

B is a peptide with a sequence containing 8 consecutive arginine residues;

with _A , _{C in} and Cm, each independently, is an amino acid residue;

M is a polyethylene glycol polymer (PEG);

□ _A and O _in are a pair of acceptor and donor fluorescent particles between which fluorescence resonant energy transfer by the Förster mechanism is possible; and where [ο _Μ -Μ] is attached in any position to X, [Oh _A -c _A | attached to any amino acid in A and [c _to -1) _in | attached to any amino acid in B, Μ is a polymer polyethylene glycol (PEG) having an average molecular weight of 500, 1000, 2000, 5000 or 10000 Da.

In one embodiment of the invention with _A , _{C in} and Cm, each independently selected from Ώ-cysteine, Oglutamate, lysine and para-4-acetyl-G-phenylalanine. c _in is the Ώ-cysteine residue.

In one embodiment of the invention, _A represents a β-glutamate or lysine residue.

In one embodiment of the invention, is a residue of para-4-acetyl-G-phenylalanine.

In one embodiment of the invention, X is capable of cleavage using matrix metalloproteinase.

In one embodiment of the invention, X contains an amino acid sequence that is capable of cleavage using ΜΜP2, ΜΜP7, ΜΜP9 or ΜΜP14. X contains an amino acid sequence selected from

RGSShAS, PbO-C (te) -AO,

KRGALU K8, Ε8ΡΑΥΥΤΑ, ORKZR, RKZR, YEREKE and R.GrP <(Ac).

In one embodiment of the invention, X comprises the amino acid sequence PbAC.

In one embodiment of the invention, X contains the amino acid sequence of PBC-C (sce) AC.

In one embodiment of the invention, X comprises the amino acid sequence of CRPAPAC8.

In one embodiment, the O _A and O _in mean Cy5 and Cy7.

In one embodiment, the O _A and O _{in the} mean Cy5 and! KOue750.

In one embodiment of the invention, O _A and O _in mean Su5 and! K0ue800.

In one embodiment of the invention, O _A and O _in mean Su5 and K'St.

In one embodiment of the invention, the molecule of formula (I) is

In some embodiments, the present invention discloses methods for visualizing target tissue in a subject in need thereof, comprising: (a) administering 8ΏΜ-14 to a subject; and (b) visualizing at least one of the imaging agents.

In some embodiments, the present invention discloses methods for visualizing target tissue in a subject in need thereof, comprising: (a) administering 8ΏΜ-15 to the subject; and (b) visualizing at least one of the imaging agents.

In some embodiments, the present invention discloses methods for visualizing target tissue in a subject in need thereof, comprising: (a) administering 8ΏΜ-23 to a subject; and (b) visualizing at least one of the imaging agents.

In some embodiments, the present invention discloses methods for visualizing target tissue in a subject in need thereof, comprising: (a) administering 8ΏΜ-24 to the subject, and (b) visualizing at least one of the imaging agents.

In some embodiments, the present invention discloses methods for visualizing target tissue in a subject in need thereof, comprising: (a) administering 8ΏΜ-25 to a subject, and (b) visualizing at least one of the imaging agents.

In certain embodiments, the present invention discloses methods for visualizing target tissue in a subject in need thereof, comprising: (a) administering 8ΏΜ-26 to the subject; and (b) visualizing at least one of the imaging agents.

In some embodiments, the present invention discloses methods for visualizing target tissue in a subject in need thereof, comprising: (a) administering 8EM-27 to a subject, and (b) visualizing at least one of the imaging agents.

In some embodiments, the present invention discloses methods for visualizing target tissue in a subject in need thereof, comprising: (a) administering 8EM-32 to a subject, and (b) visualizing at least one of the imaging agents.

In some embodiments, the present invention discloses methods for visualizing target tissue in a subject in need thereof, comprising: (a) administering 8EM-35 to a subject, and (b) visualizing at least one of the imaging agents.

According to some embodiments of the invention, targeted delivery of the imaging agent to the cell or tissue allows a medical professional to visualize / obtain an image of a particular tissue (e.g., cancerous tissue).

According to some embodiments of the invention, targeted delivery of the imaging agent to the cell or tissue allows a medical professional to remove (or surgically excise) a specific tissue (e.g., cancerous tissue).

According to some embodiments of the invention, the targeted delivery of the imaging agent to the cell or tissue allows a medical professional to remove (or surgically excise) a specific tissue (eg, cancerous tissue) while reducing the margins of the resection.

According to some embodiments of the invention, targeted delivery of the imaging agent to the cell or tissue allows the medical professional to remove (or surgically excise) the tumor / cancerous tissue and reduces the likelihood of re-emergence of the tumor / cancerous tissue.

According to some variants of the invention, the targeted delivery of the imaging agent to the cell or tissue allows a medical professional to minimize the mass of tumor / cancer tissue.

According to some embodiments of the invention, targeted delivery of the imaging agent to the breast cancer tissue reduces the likelihood of unnecessary surgeries or reoperations.

According to some embodiments of the invention, targeted delivery of the imaging agent to the cell or tissue allows the medical professional to more accurately select the target (e.g. cancer) tissue (e.g., perform a biopsy (e.g., resection biopsy, incision biopsy, aspiration biopsy, or puncture biopsy).

According to some embodiments of the invention, the targeted delivery of the imaging agent to the cell or tissue allows a medical professional to visualize / obtain an image of a particular tissue (e.g., cancerous tissue) in excised tissue containing healthy tissue. The ability to identify the target tissue (e.g., cancerous tissue) will help the pathomorphologist to determine where to cut for the assessment of pathology, and reduces the chance of missing an unhealthy tissue (e.g., cancerous tissue) and taking a healthy tissue sample, which can give a false negative result.

In some embodiments of the invention, tissue (eg, cancerous tissue) removed after imaging with a compound of formula I is used to prepare a slice or glass for pathological examination.

In some embodiments of the invention, tissue (eg, cancerous tissue) removed after imaging with a compound of formula I is used to prepare a section or glass for pathological examination to determine whether the tissue is malignant or benign.

According to some variants of the invention, the targeted delivery of the imaging agent to the breast cancer tissue allows the medical professional to accurately determine the stage of the cancer, which enables him to make a decision regarding the treatment.

According to some embodiments of the invention, the targeted delivery of the imaging agent to the cancerous tissue allows a medical professional to determine the size of the tumor (cancerous tissue) or the spread (e.g. metastatic lesion) of the cancerous tissue.

In some embodiments of the invention, targeted delivery of the imaging agent to a cell or tissue allows a healthcare professional to determine an effective treatment regimen.

According to some embodiments of the invention, the selective delivery of a molecule of formula I containing an imaging agent is used in directed surgery (imaging surgery).

According to some variants of the invention, the selectively delivered molecule is localized mainly in cancerous or other pathological tissues with increased protease activity (for example, tissues in which an inflammatory reaction is observed).

In some embodiments, the selective delivery of a molecule of Formula I containing an imaging agent is used in targeted surgeons to remove a colorectal cancer.

In some embodiments of the invention, targeted surgery using selective molecular delivery allows the surgeon to remove as little healthy (i.e., non-cancerous, non-cancerous) tissue as possible.

- 33,030,795

In some embodiments of the invention, targeted surgery using selective delivery of a molecule allows the surgeon to visualize and remove a larger volume of tissue than he could remove if there was no selective delivery of the molecule.

In some embodiments, surgery is surgery using fluorescence imaging.

Imaging agents.

In some embodiments, the imaging agent is a colorant.

In some embodiments, the imaging agent is a fluorescent particle.

In certain embodiments, the imaging agent is selected from: a fluorescent protein, a fluorescent dye, a fluorescent material, or a combination thereof.

All fluorescent particles (agents) are encompassed by the term fluorescent particle. The specific examples of fluorescent particles provided herein are illustrative and are not intended to limit the fluorescent particles for use with the targeting molecules of the present invention.

Examples of fluorescent dyes include, but are not limited to, xanthenes (for example, rhodamines, rhodols, and fluoresceins and their derivatives); Ytapek; coumarins and their derivatives (for example, umbelliferone and aminomethylcoumarins); aromatic amines (e.g. danzyl; squarates); benzofurans; fluorescent cyanines; indocarbocyanins; carbazoles; (dicyanomethylene) pyranes; polymethine; oxabenzanthran; xanthene; pyrilia; carbostyl; perylene; acridone; rubren; anthracene; coronine; phenanthracene; pyrene; butadiene; stilbene; porphyrin; phthalocyanine; chelating complexes of lanthanides; rare earth chelate complexes; and derivatives of these dyes.

Examples of fluorescein dyes include, but are not limited to, 5-carboxyfluorescein, fluorescein-5-isothiocyanate, fluorescein-6-isothiocyanate and 6-carboxyfluorescein.

Examples of rhodamine dyes include, but are not limited to, tetramethyl rhodamine-6 isothiocyanate, 5-carboxytetramethyl rhodamine, derivatives of 5-carboxyrodol, tetramethyl and tetraethyl rhodamine, diphenylmethyl and diphenylethyl rhodamine, dinaphthyl rhodamine, rhodamine 101 sulfonyl chloride АЗ (sold).

Examples of cyanine dyes include, but are not limited to, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, ΙΕΌΥΕ680, A1ex P1iog 750, 1H1) \ e800C \\ '. 1CO.

Examples of fluorescent peptides include the green fluorescent ORP protein (OGEP R1ioge8sep1 Rgo1e1n) or derivatives of OPP (for example, EvRP, EvRP2, azurite (A / IgNe), tKa1ata1, ECPR, azure (Seg1eap), cyan UREP1, CAPepe UREP.

Fluorescent labels are detected by any suitable method. For example, a fluorescence tag can be detected by exciting a fluorochrome (fluorophore, fluorescent dye) with light of the appropriate wavelength and detecting the resulting fluorescence, for example, by examining under a microscope, visually, using photographic film, using electronic detectors such as charge coupled devices (CCD, CCC photomultipliers, etc.

In some embodiments, the imaging agent is labeled with a positron-emitting isotope (e.g., ¹⁸ P) for positron emission tomography (PET, PET), a gamma-emitting isotope (e.g., ^99t Tc) for single-photon emission computed tomography (ZREST), or a paramagnetic molecule or a nanoparticle (for example, About ³⁺ chelate, or a coated magnetite nanoparticle) for magnetic resonance imaging (MRI, ME1).

In some embodiments, the imaging agent is labeled with a gadolinium chelate, iron oxide particles, superparamagnetic iron oxide particles and ultrafine paramagnetic particles, an agent containing manganese or gallium chelate.

Examples of gadolinium chelates include, but are not limited to, diethylene triamine-pentaacetic acid (ETA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (EOTA), and 1,4,7 triazacyclononan-M, No. , No.'-triacetic acid (LOTA).

According to some variants of the invention, the imaging agent is a fluorophore with absorption bands in the near infrared region for imaging in the near infrared region (near infrared spectrum), luciferase (fireflies, bacteria or intestinal cavities) or another luminescent molecule for bioluminescent imaging or vesicle (vesicle) filled with perfluorocarbon for ultrasound imaging.

In some embodiments, the imaging agent is a nuclear probe.

In some embodiments, the imaging agent is a ZREST or PET radionuclide probe.

According to some embodiments of the invention, the radionuclide probe is selected from: technetium chelate, copper chelate, radioactive fluorine, radioactive iodine and indium chelate.

Examples of Tc chelates include, but are not limited to, ΗΥΝΙΟ, ETRA and EOTA.

- 34 030795

According to some embodiments of the invention, the imaging agent comprises a radioactive particle (radioactive agent), for example a radioactive isotope such as ²¹¹ A1, ¹³¹ Ι, ¹²⁵ Ι, ⁹⁰ Υ, ^18b Ke, ¹⁸⁸ Ke, ¹⁵³ 8sh, ²¹² Βί, ³² P, ^b4 Cu, radioactive isotopes Bi et al.

Therapeutic use.

Selective delivery of the molecules of Formula I enables targeted delivery of therapeutic agents to specific cells / tissues (e.g., cancerous tissues). These molecules contain a basic peptide sequence (B) intended for transfer through the cell membrane, an acid peptide sequence (A) that inhibits the capture of peptide B in cells, a linker X that cleaves under certain conditions, therapeutic agents associated with peptides A and B , or X and a high molecular weight carrier.

In some embodiments of the invention, cleavage of linker X allows peptide B to be separated from peptide A and enables the transfer of peptide B (and any associated therapeutic agents) across the cell membrane or retention of B into tissue.

In some embodiments, the selective delivery of molecules of formula Ι provides targeted delivery of one or more therapeutic agents to a cell or tissue.

According to some embodiments of the invention, targeted delivery of a therapeutic agent to a cell or tissue allows a medical professional to treat (treat) a particular tissue.

Source materials.

Molecules of formula II are described having the structure Αι-Χι-В of Formula II, where XI is a cleavable linker;

And ₁ means a peptide whose sequence contains from 5 to 9 acidic amino acids and has a first reactive amino acid group with _A ;

Β1 means a peptide whose sequence contains from 7 to 9 basic amino acids and has a second reactive amino acid group with _c ; and

A ₁ ^ ₁ -B ₁ contains the third reactive amino acid group with _M in A ₁ or in X !;

wherein the group (fragment) with _A is able to react with the first delivery group containing E _A , the group c _in is able to react with the second delivery group containing E _in , and the group with _M is able to react with a macromolecular carrier containing M to form a molecule of the formula I.

CA, CB and SM contain orthogonally reactive functional groups.

In some embodiments, the substituents CA, CB, and CM are each independently selected from a natural amino acid or a non-natural amino acid.

According to some embodiments of the invention, with _{A, a} and _M are each independently selected from amino acid E, D amino acid, "amino acid, β-amino acid or γ-amino acids.

According to some embodiments of the invention, CA, CB and CM are each independently selected from any amino acid containing a free thiol group, any amino acid having a Ν-terminal amino group, and any amino acid with a side chain capable of forming an oxime or hydrazone group when reacted with hydroxylamine or hydrazine.

According to some embodiments of the invention, with _{A, a} and _M are each independently selected from e-cysteine, ϋ-glutamate, and lysine p-4-acetyl L-phenylalanine.

According to some variants of the invention, c _in means an amino acid containing a free thiol group.

According to some embodiments of the invention with _a mean E-cysteine.

According to some variants of the invention with _A means any amino acid having a terminal amino group.

According to some variants of the invention with _A means E-glutamate.

In some embodiments, cA is lysine.

According to some embodiments of the invention, _M means any amino acid with a side chain capable of forming an oxime or hydrazone bond when reacted with a hydroxylamino group or with hydrazine.

In some embodiments, with _M is para-4-acetyl b-phenylalanine.

The expression orthogonally reactive means that, during sequential reactions, many groups can bind to a molecule that do not cross-react with each other, which allows each group to bind specifically to the molecule in the presence of other groups. According to some embodiments of the invention, three groups (E _A , E _c , and E _M ) are able to bind to A ₁ -X ₁ -Β ₁ via _A , b and cM during three consecutive independent non-cross reactions, so that each group binds with only one site in A ₁ -X ₁ -Β ₁ .

In some embodiments, the present invention includes a molecule having an amino acid sequence

- 35,030,795 where o is 5- (amino-3-oxapentanoyl);

P ( _4-Ac ) means paraacetyl- (b) -phenylalanine and

C ( _Me ) means 8-methyl- (b) -cysteine.

According to some variants of the invention, the molecule further comprises a polymer polyethylene glycol (PEG, PEO). According to some embodiments of the invention, the PEO polymer is covalently linked to a molecule in the region of the P ( _4-Ac ) unit. According to some variants of the invention, the molecule contains groups that can react orthogonally. In some embodiments of the invention, groups that can react orthogonally are selected from amine, thiol, and acetylphenylalanine. According to some variants of the invention, the molecule contains an amino group, a thiol group and a phenylalanine group.

In some embodiments, the PEO polymer has an average molecular weight of 500 Da. In some embodiments, the PEO polymer has an average molecular weight of 2000 Da. In some embodiments, the PEO polymer has an average molecular weight of 5,000 Da. In some embodiments, the PEO polymer has an average molecular weight of 10,000 Da. In some embodiments, the PEO polymer has an average molecular weight of 20,000 Da. In some embodiments, the PEO polymer has an average molecular weight of 40,000 Da. In some embodiments, the present invention includes the use of a given molecule in the synthesis of a compound (molecule) of formula Ι.

In some embodiments, the present invention provides a molecule having an amino acid sequence

where all the glutamate and arginine residues are residues of B-amino acids; o means 5- (amino-3-oxapentoyl);

C ( _te ) means 8-methyl- (T) -cysteine;

PEO ( _3K ) means α-amino-y-amide poly (ethylene glycol) having an average molecular weight

3000 Yes.

According to some variants of the invention, the molecule further comprises a fluorescent particle. In some embodiments, the present invention includes the use of a given molecule in the synthesis of a compound (molecule) of formula Ι.

- 36,030,795

- 38 030795

- 41 030795

Examples

Materials and methods.

Chromatographic (HPLC) pure acetonitrile was obtained from p! 8Let Zsleprls (РЫШрз- 42 030795

Wagu, RA). Purified water was collected using a Μίίίί-воды water purification system (MlShroge, VesP'ogs), MA). P £ p (pentafluorophenyl) ester of 3-maleimidopropionic acid was obtained from Mo1Ci1A1 Wuzshepses (VoiGe, CO). ZFR-EDTA buffer was obtained from Tekpoua (NoshzTeg, SA). Trifluoroacetic acid (TPA), dimethylformamide (SUP) and Ν-methylmorpholine (ΝΜΜ) were obtained from the company §1§ta-A1yg1cb (Μ Poikike, Sh1). α-Mercaptoethyl-U-methoxy polyoxyethylene | Μ «(ΜΜ) 2000, 5000, 20,000 and 40,000) [TREO (2K) -8H, TREO (5K) -8H, TREO (20K) -8H, TREO (40K) -8H ] and α-aminoxyl-U-methoxy polyoxyethylene | Μ «(ΜΜ) 2000, 5000, 20,000 and 40,000) [TREO (2K) -OYAN ₂ , TREO (5K) -OYAN ₂ , TREO (20K) -OYAN ₂ , TREO (40K) -OYAN ₂ ] was obtained from the company NOR Ateps SoOOogayop (Rute, CA). TRRO (1K) -X'1 ^; 1X'112 received from Haposs (X'e \ u Wogk). GK-Bue 800SSh maleimide (Μа1-IК ^ уе) and GKBue 750 succinimidyl ether were obtained from С-Sog Vyushepses (Btso1p, XE). Lyophilized peptides P1-P17 were prepared using conventional peptide coupling methods on a standard resin.

LC-MS analysis was performed on an Adiep! 1200 model 8B in combination with an HP 8СГЕХ АР1 3200 HPLC / MS / MS system, equipped with an autosampler (automatic high-speed dispenser) STS RAB operating at 4 ° С, a vacuum degasser, a binary pump, and U-U18 detector, corresponding to the analytical program Apa1uz1 1.5 and with a RRepotepech column (Kte1ex 2.6 cS18 100 A, 100x2.1 mm), or on a Sha1egz 2695 separation module equipped with a two-wave absorption detector \ Wa1egz 2487 in combination with a RTschap Ι-C'O Bes XP mass spectrometer. The device is equipped with analytical software (software) XaRbig and a Reeke 8Spys column (Trap 200 5 μm, C18-MS, 50x2.1 mm).

HPLC was performed on an Adyep system! (Adyep! 1200 series) and on the Tregoto 8 Shepys column (NuregzR Oo1P C18, 5 μm, 250x10 mm), or on a preparative HPLC \ Wa1egz BeRa Rger system and on the Wapap column (P75B, C18, 15 μm, 1200 g), or on the system \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\uality One At One Equipped With Two-Wave Absorption Detector Reprotepech (1ipa, C18 (2), 5 μm, 100 AX 150x30 mm). The mobile phase consisted of water (0.05% TPA) (solvent A) / acetonitrile (0.05% TPA) m (solvent B) gradient.

Centrifugation was carried out at 4 ° C in a ErrepRog! Centrifuge! 5810K or Wesstap Μ ^ s ^ oίiβе®

eighteen.

Typical materials for the synthesis of molecules for selective delivery of the present invention include, but are not limited to, any of the peptides P-1, P-2, P-3, P-4, P-5, P-6, P-7, P- 8, P-9, P-10, P-11, P12, P-13, P-14, P-15, P-16 and P-17.

The above starting materials are listed below in the table

Peptide Sequences Peptide R-1 eeeeeeeeeRROS (me) ASgptptpts Peptide R-2 eeeeeRROS (me) ASgptggpts Peptide R-3 Eeeeeer (4-Ac) ORROS (Me) AOptptgs Peptide R-4 eeeeeeeer (4-Ac) ORROS (me) ASgptptpts Peptide R-5 (As) eeeeeRROS (me) ASgptgggptsk Peptide R-6 eeeeeRROS (me) ASgoR ( _4- ac) Ptggps Peptide R-7 eeeeeeeeeRROS (me) ASgptptptptsoR (4-Ac) Peptide R-8 [TREO (2K)] cttptgOFRSgS (me) ASgoyeeek Peptide R-9 [tRESg (5K)] cttptgOFRSgS (me) ASgoyeeek Peptide R-10 eeeeeRROS (me) AOtgptpts [RESg (zk)]

Abbreviations.

Standard 1 letter abbreviations were used for all sequences. The lower characters indicate B-amino acids. All peptides were amidated at the C-terminus.

o: 5- (amino-3-oxapentanoyl);

P (4-aC): paraacetyl- (B) -phenylalanine;

C (shu 8-methyl- (B) -cysteine.

REO (3K): α-amino-U-amide polyethylene glycol with an average molecular weight of 3000 Da; TREO (2K): α-carboxy-Щ-methoxy polyethylene glycol with an average molecular weight of 2000 Da;

- 43 030795 treo (5k): a-carboxy-y-methoxy polyethylene glycol with an average molecular weight of 5000 Da.

Ac: acetyl.

In the schemes presented in the examples below, b means h; t \ 1 means mmol; O1uste means glycine; apshie means aniline.

To a solution of the peptide P-1 (8 mg, 2.1 μmol) in ΌΜΡ (0.8 ml) at room temperature, 1 Кеуе 800СШ maleimide (2 mg, 1.7 μmol) and Ν-methylmorpholine (10 μl, 91 μmol) were added with stirring in the dark. The progress of the reaction was monitored by LC-MS, usually the reaction was completed after 1 h. The mixture was used directly in the next step without further purification.

To the reaction mixture obtained above, 3-maleimidopropionic acid Ρίρ-ester (2 mg, 6.0 μmol) was added. The resulting mixture was stirred at room temperature in the dark for 20 hours. Purification by RP-HPLC (RP (reverse phase) -HPLC) gave Intermediate 5 (2.1 mg, 22% based on two steps). Calculated: [M + 3H] ³⁺ (^ ₈ 7Η ₂₉₀ Ν ₅₉ Ο ₆₄ δ ₆ ) t / ζ = 1526; Found: ΕδΙ: [M + 3H] ³⁺ (C187H29 <No. 9Ob48b) t / ζ = 1526.

Molecule synthesis for selective delivery of 8ΌΜ-2.

A mixture of intermediate 5 (1.5 mg, 0.27 μmol) and TREO (40K) -8H (10 mg, 0.25 μmol) in ZFREDTA buffer (0.5 ml, 137 mM ZhS1, 7 mM Ν α ₂ ΗΡΟ ₄ , 3 mM KS1, 1.4 mM K ₃ PO ₄ , 4 mM EDTA, pH 7.4) was stirred at room temperature in the dark for 20 hours. Purification by RP-HPLC gave the molecule for selective delivery of 8ΌΜ-2 (7.0 mg, 61%).

Molecules for the selective delivery of δΌΜ-1, δΌΜ-3, 8ΌΜ-4 and 8ΌΜ-5 were obtained analogously to 8ΌΜ-2 from peptide P-1.

Example 2. Synthesis of δΌΜ-6 from peptide P-2

- 44 030795

Synthesis of Intermediate 7.

To a solution of peptide P-2 (378.5 mg, 0.1 μmol) in OMR (25 ml) with stirring at room temperature in the dark, Cy5 maleimide (87 mg, 0.09 μmol) and Ν-methylmorpholine (350 μl, 3.2 μmol) were added. The progress of the reaction was monitored by LC-MS, usually the reaction was completed after 1 h. The mixture was used directly in the next step without further purification.

To the reaction mixture obtained above was added P £ p-ester of 3-maleimidopropionic acid (50 mg, 0.15 μmol). The resulting mixture was stirred at room temperature in the dark for 5 hours. Purification by RP-HPLC afforded intermediate 7 (108 mg, 27% in two steps). Calculated: [M + 2H] ² + (С148Н23 ₅ Д ₅ 1О4484) t / ζ = 1780; Found E81: [M + 2H] ²⁺ t / ζ = 1780.

Molecular Synthesis for Selective Delivery of 8CM-6.

A mixture of intermediate 7 (95 mg, 21.2 μmol) and TREO (40K) -8H (0.9 g, 22.5 μmol) in ZFREDTA buffer (40 ml, 137 mM DaC1, 7 mM Yes ₂ NRA ₄ , 3 mM KC1, 1.4 mM K ₃ PO ₄ , 4 mM EDTA, pH 7.4) was stirred in the dark at room temperature for 20 hours. Purification by RP-HPLC gave a molecule for selective delivery of 3ΏΜ-6 (0.85 g, 90%).

Molecules for the selective delivery of 8OM-7 and 8OM-8 were obtained similarly to 8OM-6 from peptide P-2.

Example 3. Synthesis of 3ΏΜ-25 from peptide P-3

- 45,030,795

Synthesis of Intermediate 8.

To a solution of the peptide P-3 (200 mg, 49.6 μmol) in ΌΜΡ (5 ml) with stirring at room temperature in the dark, Su5 maleimide (60 mg, 65.6 μmol) and Ν-methylmorpholine (80 μl, 0.73 μmol) were added. The progress of the reaction was monitored by LC-MS; it was completed after 1 h. Ether (40 ml) was added to the mixture. The precipitate was obtained by centrifugation, washed with ether (40 ml X 2) and purified using HPLC (HPLC), received the intermediate 8 (141 mg, 61%). Calculated: [M + 3H] ³ + (^ 52Η242Ν51Ο4384) t / ζ = 1200; Found E81: [Μ + 3Η] ³ + (С152Н ₂₄ ^ ₅₁ О ₄₃ 8 ₄ ) t / ζ = 1200.

Synthesis of Intermediate 9.

To a solution of intermediate 8 (101 mg, 21.8 μmol) in ΌΜΡ (10 ml) at room temperature were added succinimidyl ester of Cy7 carboxylic acid (40 mg, 41.1 μmol) and Ν-methylmorpholine (0.2 ml, 1.8 μmol). The resulting mixture was stirred at room temperature in the dark for 36 hours. Ether (35 ml) was added to the mixture. The precipitate was collected after centrifugation and washed with ether (40 ml X 2). By purification of the mixture by RP-HPLC, intermediate 9 (28.1 mg, 25%) and intermediate 8 (63 mg) were obtained. Calculated: [M + 3H] ³ + (^ 7 ^ 2 ^ 305086) t / ζ = 1421; Found E81: [M + 3H] ³ + (^ 7 ^ 2 ^ 305086) t / ζ = 1421.

Molecular Synthesis for Selective 8ΌΜ-25 Delivery

A mixture of intermediate 9 (28.1 mg, 5.4 μmol) and tΡEΟ (2Κ) -NΟ ₂ (17 mg, 7.6 μmol) in glycine buffer (4 ml, 0.1 M, 20 mM aniline, pH 3.0) and acetonitrile (0.8 ml) was mixed in darkness at room temperature for 24 hours. At the end of the reaction, acetophenone (10 μl, 86 μmol) was added. The mixture was stirred at room temperature for 2 hours. Purification by RP-HPLC gave a molecule for selective delivery of 8ΌΜ-25 (25 mg, 63%).

Molecules for the selective delivery of 8ΌΜ-9, 8ΌΜ-10, 8ΌΜ-22, 8ΌΜ-23, 8ΌΜ-24, 8ΌΜ-26, 8ΌΜ27, 8ΌΜ-29 and 8ΌΜ-31 were obtained similarly to 8ΌΜ-25 from peptide P-3.

Example 4. Synthesis of 8ΌΜ-15 from peptide P-4

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Synthesis of Intermediate 11.

To a solution of P-4 peptide (30 mg, 6.2 μmol) in ΌΜΡ (2 ml) with stirring in the dark at room temperature were added Cy5 maleimide (7.5 mg, 8.2 μmol) and Ν-methylmorpholine (15 μl, 0.14 μmol). The reaction was monitored by LC-MS and completed after 1 h. The mixture was purified by HPLC to give intermediate 11 (19.7 mg, 59%). Calculated: [M + 3H] ³⁺ (С178Н282 ^ 9О5634) t / ζ = 1424; Found E81: [M + 3H] ³ + (C178H ₂₈₂ ^ ₉ O ₅₆ 8 ₄ ) t / z = 1424.

Synthesis of Intermediate 12.

To a solution of intermediate 11 (15 mg, 2.8 μmol) in ΌΜΡ (1.5 ml) at room temperature were added succinimidyl ester Cy7 carboxylic acid (4 mg, 4.3 μmol) and Ν-methylmorpholine (10 μl, 91 μmol). The resulting mixture was stirred in the dark at room temperature for 48 hours. Purification by RP-HPLC gave intermediate 12 (5.0 mg, 30%). Calculated: [M + 3H] ³ + (C213H322C, 1O63 36) t / ζ = 1645; Found E81: [M + 3H] ³ + (C213H ₃₂ ^ ₆₁ O ₆₃ 8 ₆ ) t / z = 1645.

Molecule synthesis for selective delivery of 8ΌΜ-15.

A mixture of intermediate 12 (1.1 mg, 0.18 μmol) and TPEC (2K) -OKH ₂ (1 mg, 0.5 μmol) in glycine buffer (1 ml, 0.1 M, 20 mm aniline, pH 3.0) and acetonitrile (0.2 ml) was stirred at room temperature in the dark for 1 day. Purification by RP-HPLC provided a molecule for the selective delivery of 8ΌΜ-15 (0.6 mg, 42%).

Molecules for the selective delivery of 8ΌΜ-11, 8ΌΜ-12, 8ΌΜ-13, 8ΌΜ-14 and 8ΌΜ-28 were obtained analogously to 8ΌΜ-15 from intermediate 11.

Example 5. Synthesis of 8ΌΜ-16 from peptide P-5

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O ^ OH OH, “OH SK.ON

О · 'Н С - О''нГЛ' ^Λ Ν> Τ ^Ν γ ^ Ν ' ⁴ π- ^Ν ' _Ι > Ν '^< γ ^Ν ^ Ον ^Ν γ.

^N ° 1 ^N ° 1 ^N ° ⁰ ° ⁿ θ ι ^{Η 5} Squeegee sHon ^M

Η ₂ Νψ \ ΙΗ Η ₂ ΝγΝΗ Η ₂ Νψ \ ΙΗ Η ₂ Ν ^ ΝΗ ΗΝ _Υ ΗΝ H1 \ to H1 \ to νη ₂

-Λνη η Ο ^{ύ Ί} BUT Η ο> Η Ο 1 Η ο> Η Ο | Η ο>, Χ ^ν ΧνΧ ^ν Χν ^ Γ ^Ν ίΛν ^λ υ ^ν ίΛν ^λ υ ^ν Χν ^λ υ ^ν Χν ^λ υ ^ν ΧνΥτ ^ι

Χδ But ^ NokNokNokNokNO Val on kn ι 4ιυ ki 4ιυ

R-5

ΝΗ

ΗΝ% Η ₂

ΗΝΗΜΗ,

NN

ΗΝ% Η ₂ 'ΝΗ ηγΛνη,

Su5-Ma1 ΝΜΜ, ϋΜΡ, 1 ή

ΟγΟΗ ΟγΟ Η Ο Γ Η Ο Γ Η

ΟγΟΗ

Η

Ν% ^Ν γ ^Λ Ν% ^Ν γ ^Α Ν ^Λ τ ^Ν ^ ο ^ τ ^Ν Ϋ

Η ό to Η ό o> he α η ό ΌΗ ο ₀ + Ν _Η η 0 ^& Ί Η ο Η Ο Λ Η ο 73 Η Ο ^! Η O to Η O to Η

Η ₂ ΝγΝΗ Η, Ν ^, ΝΗ Η ₂ Ν _γ ΝΗ Η ₂ Ν ^ ΝΗ ΗΝ ^ ΗΝ ^ ΗΝ ^ ΗΝ.

TTL

Ο

Η ΟΊ Η ο. ..

ΝΓΑ, ^^ Ν.Λ. _Ν - _γ Ν Η Ο

ΝΗ ΝΗ

ΗΝ ^ ΝΗ ₂ ην% η ₂

Ν> Γ Η ο

ΝΗ

ΗΙψ ΝΗ ₂

<\ Ρ> Υν ην ^ νη ₂ λ;

ο

5Ο ₃ Η

ΟΡίρ

ΝΜΜ, EMR, 20 ή

ΟγΟΗ

Ο Γ Η Ο

ΛΧ

Η ο ν ^ υ ^ν · η ο, ΟΗ ΟγΟΗ Η Ρ Γη

Η ο 0> 0Η

Η ₂ Ν _γ ΝΗ Η ₂ ΝγΝΗ Η ₂ Ν _γ ΝΗ Η ₂ ΝγΝΗ ΗΝ., ΗΝ. ΗΝ ΗΝ.

Γ Ύ

ΗΝ '' γΤΝΗ Η Ο ^{ύ Ί} Η ο Η Ο = Η ο Λ Η Ο Τ Η ρ Τ η ο λ .. · ^Ι γ ^Ν '7 ^ Ν'γ ^Ν Λ ^Α Ν ^ Γ ^Ν γ ^ Ν'> Γ ^Ν γ ^ Ν ^ ΓΝγΛΝ ^ _γ Νγί _ν Α ^ ΝγΧ _Ν ΛγΝ η ₂

Χο but ^{in the} knock-down button _z ϊ ϊ 7 ^Η

NING ΗΝ% Η ₂ ΗΝ ^Λ ΝΗ2 ΗΝ ^Λ ΝΗ2

5Ο ₃ 3Ο ₃ Η ТРЕС (40К) -5Н ρΗ 7.4, ΡΒ5, 20 ή

ΟγΟΗ ΟγΟΗ ΟγΟΗ о Гн о Гн о Гн '\ - ^λ ν..ν

Η Ο ¹ to ⁰

Un Ο '^ ΌΗ

Η ° Ο (-Λνη η SLO Η ρ, ΥΛΥ> Ν ^ 'Λο Η Ο ⁵ Η Ο _ Μ \ ν = 40,000 5Ο <

ΟΧ '

Η ₂ Ν.ΝΗ Η ₂ ΝγΝΗ Η ₂ Ν.ΝΗ Η ₂ Ν _γ ΝΗ ίη Λ

NSC ΗΝ. ΗΝ ΗΝ. (Ρ3 ^ Ρρζ

I \ and ίΊ \ ι_ι Γ \ k

5ΌΜ-16

ΧΗ ΛΙΗ to Ί4Η kk. ην% ιη ₂ ην ^λ νη2 ην ^λ νη2 ην ^λ νη ₂ 4

3Ο ₃ Η

Synthesis of Intermediate 20.

To a solution of the P-5 peptide (20 mg, 5.2 μmol) in ΏΜΕ (1 ml) with stirring in the dark at room temperature were added Cy5 maleimide (6 mg, 6.6 μmol) and Ν-methylmorpholine (12 μl, 109 μmol). The progress of the reaction was monitored by LC-MS and usually completed after 1 h. The mixture was used directly in the next step without further purification.

To a solution of the mixture obtained above in ΩΜΡ (1 ml) at room temperature was added the Ρίρ-ester of 3-maleimidopropionic acid (2.5 mg, 7.5 μmol). The resulting mixture was stirred at room temperature in the dark for 20 hours. Purification by RP-HPLC afforded intermediate 20 (7.3 mg, 30% in two steps). Calculated: [M + 3H] ³⁺ (Χ5 ₆ Η ₂ 5 ₀ Ν ₅₃ 0 ₄₆ 8 ₄ ) t / ζ = 1244; Found Ε5Ι: [M + 3H] ³⁺ Χ156Η250Ν5304684) t / ζ = 1244.

Molecule synthesis for selective delivery of 8ΏΜ-16.

A mixture of intermediate 20 (1.4 mg, 0.3 μmol) and tΡΕC (40Κ) -8Η (14 mg, 0.35 μmol) in ZFREDTA buffer (2 ml, 137 mM ΝαΟ, 7 mM Να ₂ ΗΡ 0 ₄ , 3 mM KC1, 1.4 mM Κ ₃ Ρ0 _4, 4 mM EDTA, ρΗ 7.4) was stirred at room temperature in the dark for 20 h. Purification by RP-HPLC gave molecule to selectively deliver 8ΏΜ-16 (6.5 mg, 49%).

Molecules for the selective delivery of 8ΏΜ-17, 8ΏΜ-18 were obtained analogously to 8ΏΜ-16 from peptide P-5.

Example 6. Synthesis of 8ΏΜ-33 from peptide P-7

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Synthesis of Intermediate 21.

To a solution of P-7 peptide (20 mg, 4.1 μmol) in ΌΜΡ (1 ml) with stirring at room temperature in the dark were added Cy5 maleimide (6 mg, 6.6 μmol) and Ν-methylmorpholine (10 μl, 91 μmol). The progress of the reaction was monitored by LC-MS and the reaction was completed after 1 h. The mixture was purified by RP-HPLC to give intermediate 21 (9 mg, 40%). Calculated: [M + 3H] ³ + (^ ₈₂ Η ₂₈₉ Ν ₆₀ Ο ₅₈ δ ₄ ) t / ζ = 1458; Found ΕδΙ: [Μ + 3Η] ³ + ((78: 11 ^ 60 (^.,) T / ζ = 1458.

Synthesis of Intermediate 22.

To a solution of intermediate 21 (9 mg, 1.6 μmol) in ΌΜΡ (1 ml) at room temperature were added succinimidyl ester of Cy7 carboxylic acid (3 mg, 3.1 μmol) and Ν-methylmorpholine (10 μl, 91 μmol). The resulting mixture was stirred in the dark at room temperature for 24 hours. Purification by RP-HPLC gave Intermediate 22 (4.9 mg, 50%). Calculated: [M + 3H] ³ + ^ 217Η329Ν62θ65δ6) t / ζ = 1679; Found ΕδΙ: [Μ + 3Η] ³ + (^ 17Η ₃₂ 9Ν ₆₂ Ο ₆ 58 ₆ ) t / ζ = 1679.

Molecule synthesis for selective delivery of δΌΜ-33.

A mixture of intermediate 22 (0.9 mg, 0.15 μmol) and TREO (10K) -OHI ₂ (3 mg, 0.3 μmol) in glycine buffer (1 ml, 0.1 M, 20 mM aniline, ρ 3.0) and acetonitrile (0.2 ml) were mixed in darkness at room temperature for 3 days. Upon completion of the reaction, acetophenone (10 μl, 86 μmol) was added. The mixture was stirred at room temperature for 2 hours. Purification by RP-HPLC afforded the molecule for selective delivery of δΌΜ-33 (0.8 mg, 38%).

The molecule for selective delivery of δΌΜ-34 was obtained similarly to δΌΜ-33 from intermediate 22.

Example 7. Synthesis of δΌΜ-36 from peptide P-8

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Synthesis of Intermediate 23.

To a solution of the peptide P-8 (10 mg, 1.7 μmol) in PMP (1 ml) with stirring in the dark at room temperature were added Cy5 maleimide (4 mg, 4.4 μmol) and Ν-methylmorpholine (10 μl, 91 μmol). The reaction mixture was stirred at room temperature for 1 hour. Purification by HPLC gave Intermediate 23 (5.4 mg, 48%).

Molecular Synthesis for Selective Delivery 81) M-36.

To a solution of intermediate 23 (5.4 mg, 0.82 μmol) in EMR (1 ml) at room temperature were added succinimidyl ester of Cy7 carboxylic acid (3 mg, 3.1 μmol) and Ν-methylmorpholine (10 μl, 91 μmol). The resulting mixture was stirred at room temperature in the dark for 36 hours. Purification by RP-HPLC afforded 8RM-36 (0.7 mg, 13%).

Molecules for the selective delivery of 8PM-37 were prepared analogously to 8PM-36 from peptide P-8.

Example 8. Synthesis of 8RM-38 from peptide P-10

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Synthesis of Intermediate 24.

Cy5 maleimide (4 mg, 4.4 μmol) was added to a solution of P-10 peptide (10 mg, 1.4 μmol) in PBS buffer (pH 7.4, 1 ml) with stirring in the dark at room temperature. The reaction mixture was stirred at room temperature for 1 h. Purification by RP-HPLC afforded intermediate 24 (7.9 mg, 79%).

Molecular Synthesis for Selective Delivery 8ΟΜ1-38.

To a solution of intermediate 24 (7.9 mg, 1.1 μmol) in ΏΜΡ (1 ml) at room temperature were added succinimidyl ester of Cy7 carboxylic acid (2 mg, 2.0 μmol) and Ν-methylmorpholine (10 μl, 91 μmol). The resulting mixture was stirred in the dark at room temperature for 36 hours. Purification by RP-HPLC provided molecules for the selective delivery of 8ΟΜ-38 (1.7 mg, 19%).

Molecules for the selective delivery of 8ΟΜ-39 were obtained similarly to 8ΟΜ-38 from peptide P-10.

Example 9. Synthesis of 8ΏΜ-40 from peptide P-8

δϋΜ-40

Synthesis of Intermediate 25.

To a solution of P-8 peptide (10 mg, 1.7 μmol) in ΏΜΡ (1 ml) with stirring at room temperature in the dark, Su7 maleimide (4 mg, 4.2 μmol) and Ν-methylmorpholine (10 μl, 91 μmol) were added. The reaction mixture was stirred at room temperature for 1 hour. Purification by HPLC gave intermediate 23 (3.1 mg, 28%).

Molecule synthesis for selective delivery of 8ΟΜ-40.

To a solution of intermediate 23 (3.1 mg, 0.47 μmol) in ΏΜΡ (1 ml) at room temperature was added succinimidyl ester Cy5 carboxylic acid (2 mg, 2.1 μmol) and Ν-methylmorpholine (5 μl, 46 μmol). The resulting mixture was stirred in the dark at room temperature for 24 hours. Purification by RP-HPLC afforded 8ΏΜ-40 (1.4 mg, 41%).

Example 10. Synthesis of 8ΟΜ-30 from peptide P-3

- 51 030795

A mixture of intermediate 8 (3 mg, 0.64 μmol) and Su7-NH ₂ (3 mg, 2.9 μmol) in glycine buffer (4 ml, 0.1 M, 20 mm aniline, pH 3.0) and acetonitrile (0.1 ml) was stirred in the dark at room temperature temperature for 36 hours. Purification by RP-HPLC gave intermediate 26 (1.1 mg, 31%). Calculated: [M + 3H] ³ + (С189Н28 ^ 55О50§6) t / ζ = 1441; Found E8P [M + 3H] ³ + (^ 89 ^ 8 ^ 505086) t / ζ = 1441. Su7ΟΝΗ _{2 was} obtained from Cy7-COOH and 2 - ^ - phthalimido- (aminooxy)] ethylamine under standard amide bond formation conditions followed by removal of the phthalimide protecting group with hydrazine. 2- [Νphthalimido- (aminooxy)] ethanamine was obtained from the commercial Ν-Boc-ethanolamine and Ν-hydroxyphthalimide by the Mitsunobu reaction with the removal of the Boc group using TPA.

Synthesis of Intermediate 27.

To a solution of intermediate 26 (1.1 mg, 0.2 μmol) in EMR (1 ml) at room temperature were added 3-maleimidopropionic acid PPP ester (0.5 mg, 1.5 μmol) and Ν-methylmorpholine (5 μl, 45 μmol). The resulting mixture was stirred in the dark at room temperature for 36 hours. Purification by RP-HPLC gave intermediate 27 (0.8 mg, 75%). Calculated: [M + 3H] ³ + (C1 ₉₆ H ₂₉ ^ ₅₆ O ₅₃ 8 ₆ ) t / ζ = 1491; Found E8P [M + 3H] ³ + (Cx96H29M6O53§6) t / ζ = 1491.

Molecule synthesis for the selective delivery of 8EM-30.

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A mixture of intermediate 27 (0.7 mg, 0.15 μmol) and TREO (10K) -8H (3 mg, 0.3 μmol) in ZFREDTA buffer (0.5 ml, 137 mM DaC1, 7 mM Yes ₂ NRA ₄ , 3 mM KC1, 1.4 mM K ₃ PO ₄ , 4 mM EDTA, pH 7.4) was stirred at room temperature in the dark for 40 hours. Purification by RP-HPLC provided the molecule for selective delivery of 8BM-30 (1.2 mg, 23%).

Molecules for the selective delivery of 8BM-32 and 8BM-35 were obtained analogously to 8BM-30 from peptides P-3 and P-4.

To a solution of peptide P-6 (30 mg, 7.6 μmol) in BMR (2 ml) at room temperature and with stirring in the dark were added Cy5 maleimide (9 mg, 9.4 μmol) and Ν-methylmorpholine (15 μl, 137 μmol). The progress of the reaction was monitored by LC-MS and the reaction was completed after 1 h. Purification by RP-HPLC gave intermediate 28 (24.9 mg, 68%). Calculated: [M + 3H] ³ + (C ₁₅₆ H ₂₄₉ D ₅₂ O ₄₅ 8 ₄ ) t / ζ = 1233; Found E81: [M + 3H] ³ + (C15bH249D52O4584) t / ζ = 1233.

Synthesis of Intermediate 29.

To a solution of intermediate 28 (17.7 mg, 3.7 μmol) in BMR (1.5 ml) at room temperature were added succinimidyl ester of Cy7 carboxylic acid (5 mg, 5.5 μmol) and Ν-methylmorpholine (20 μl, 0.18 μmol). The resulting mixture was stirred at room temperature in the dark for 30 hours. Purification of the mixture by RP-HPLC gave Intermediate 29 (7.1 mg, 35%). Calculated: [M + 3H] ³⁺ (Cx91H289D54O5286) t / ζ = 1455; Found E81: [M + 3H] ³ + (Cx9xH2 ₈ 9D54O5286) t / ζ = 1455.

Molecular Synthesis for Selective Delivery of 8BM-21.

A mixture of intermediate 29 (1.8 mg, 0.33 μmol) and TREO (10K) -ODH ₂ (4 mg, 0.4 μmol) in glycine buffer (1 ml, 0.1 M, 20 mm aniline, pH 3.0) and acetonitrile (0.1 ml) were mixed in darkness at room temperature for 3 days. Purification by RP-HPLC provided the molecule for the selective delivery of 8BM-21 (1.0 mg, 20%).

Molecules for the selective delivery of 8BM-19 and 8BM-20 were obtained analogously to 8BM-21 from intermediate 29.

Example 12. Enzyme-dependent enhancement of fluorescence and color change.

The molecule for selective delivery of 9 was dissolved in TSDV buffer (50 mM TPZ, pH 7.5, with 10 mM calcium chloride, 150 mM sodium chloride and 0.05% ON 35) at room temperature to give a solution with a concentration of 1 cM. Fluorescence spectra were recorded on a P-2500 fluorescence spectrometer. The Cy5 fluorescence donor was excited using light with a wavelength of 625 nm, and emission (emission) was recorded in the wavelength range from 660 to 800 nm. The maximum emission (peak of emission) of the Su5 donor is observed at ~ 670 nm, and the REET peak of the Su7 acceptor radiation is observed in the region of ~ 780 nm, as shown in FIG. 2. Peptide cleavage was initiated by adding matrix metalloprotease-2 (MMP-2) to a final concentration of 1 nM. The cleavage reaction was completed after 2 hours, and the fluorescence spectrum indicated a violation of REET (fluorescence resonant energy transfer) and a large 8-fold increase in the emission (emission) of the Cy5 donor and a 2-fold decrease in the emission of Cy7. In fact, the decrease in the inherent fluorescence of Su7 is greater, but it overlaps with the shoulder of the long waves of Su5. This result indicates that ZEM-9 induces efficient energy transfer from Cy5 to Cy7 in the intact peptide.

Example 13. Enzyme-dependent enhancement of fluorescence and color change.

A molecule for selective delivery of 10 was dissolved in TCNΒ buffer (pH 7.5) at room temperature to give a solution with a concentration of 1 mM. Fluorescence spectra were recorded on a P-2500 fluorescence spectrometer. The Cy5 fluorescence donor was excited with light at a wavelength of 625 nm and the emission (emission) was measured at 669 nm. Peptide cleavage was initiated by adding MMP-9 to a final concentration of 1 nM. The cleavage reaction was completed after 2 hours, fluorescence was increased more than 100 times after cleavage of the protease, FIG. 3. A significant increase in fluorescence (reaction) shows that the dye-quencher effectively quenches Cy5 fluorophore in the unsplit ZOM-10 molecule.

Example 14. Fluorogenic reaction when using homogenate of tumor tissue.

NT1080 cells (Ca1. # SSR-121; Atepsap Ture Cu1! Ig Co11ec! Ju, UA, iZA) were grown under conditions of the exponential growth phase in humid air with 5% CO ₂ at 37 ° C until a 80-100% monolayer was reached, and then collected for implantation in mice. Each nude mouse (a nude mouse with a piye mutation) was held by hand and each mouse was injected with 2x10 ⁶ HT-1080 cells in the adipose tissue of the mammary gland using a 25-O needle. HT-1080 tumor tissues were harvested when they reached a size of 100-200 mm ³ (usually 1-2 weeks after tumor cell implantation).

Tumor NT-1080 tissues were homogenized, destroying them using ultrasound. 1 nM MMP-9 or 10 μl of tumor tissue homogenates (TH2 and TH3) was mixed with 1 μM ZEM-10 in 100 μl of buffer for 24 hours at 37 ° C. A molecule for selective delivery of 6 was used as a fluorescence control, similar in size to the intact ZEM-10. Samples were applied to a polyacrylamide gel and separated by electrophoresis. The results are shown in FIG. 4, they show that the ZEM-10 molecule is practically not fluorescent before cleavage. After incubation with the homogenates of the tumor tissue NT-1080, the ZEM-10 is cleaved and becomes highly fluorescent. OM6001 is a common broad-spectrum MMP3 inhibitor. The fact that OM6001 inhibits cleavage indicates that cleavage of the homogenate is caused by MMPs associated with tumors.

Example 15. 1p U1yo visualization of the fluorescence contrast of the tumor.

An HT-1080 tumor xenograft model was obtained as described in Example 14 and used to evaluate the ability of the molecules to provide T1U0 fluorescence contrast of the tumor compared to surrounding tissues. Fluorescent conjugates were tested on mice carrying NT-1080 tumor to achieve a tumor size of 100-200 mm ³ (usually 1-2 weeks after implantation of tumor cells). The mobility of awake mice bearing NT-1080 tumors was limited by using a rotating tail injector (Ca!. # ET1; vat! Ga ZschepIIc, MA, IZA) and the test compound was administered intravenously (into the tail vein) of each mouse at a dose of 0.1 to 5 nmol in 100 μl of saline. When using the preparation for visualization, mice were subjected to weak anesthesia with a mixture of ketamine / xylazine (Ca!. # K-113; Z1§ta, A1ypsy, MO, IZA) by intraperitoneal injection (1 μl / g body weight) to minimize mobility .

Serial total imaging (including tumor) was performed using a complete animal fluorescence imaging system or a fluorescence microscope based on an O1utriz stereo microscope. Mice were placed on their back and visualization was performed starting from above to obtain an image from the side of the abdominal cavity of the animal. The wavelengths of excitation and emission (radiation) were chosen taking into account the dye used. Contrast was calculated by the following equation:

Contrast = (Tumor fluorescence intensity - tissue fluorescence intensity on the opposite side of the breast) / tissue fluorescence intensity on the opposite side of the breast).

A contrast value of more than 0.4 in the animal’s body as a whole is easily detected by eye in the image of the whole animal and is a good contrast. Contrast> 0.7 is high contrast.

Images of mice are obtained several times within 1-24 hours after injection.

Typical imaging results two hours after dosing the molecule for selective delivery to 6 different mice are shown in FIG. 1. In this particular case, the average value

- 54,030,795 contrast 1.1. Other compounds were tested in a similar way and the contrast values are presented in table. one.

Table 1

Brief information on the ΐη νΐνο peptide conjugate contrast values obtained on the NT-1080 xenograft model

Molecule for selective delivery Maximum contrast (> 0.4 = good;> 0.9 = high) Time to maximum contrast (hour) (very fast <4; 4 <fast <12;> 12 slowly) one Good Quickly 2 Tall Quickly 3 Tall Slow four 5 6 Tall Very fast 7 Tall Slow 8 Good Very fast 9 Tall Quickly

Example 1b S νΐνο distribution and compounds with increased accumulation in tissues.

To determine the total accumulation of dye in various organs of mice with NT-1080 xenografts, they were sacrificed and tissue samples from the blood, liver, and kidneys of the tumor were taken b hours after the compounds were introduced into the tail vein. 3-4 mice were used for each experimental point. Blood samples were stored at 4 ° C. overnight, and then centrifuged at a rotor speed of 15,000 rpm to separate serum. The organs were mixed in the RgoC buffer (0.25 mg / ml Prgoc, 0.1 mg / ml DNAase, 150 mM No. С1, 10 mM Ττΐδ pH 8.0, 0.2% δϋδ) in a volume of 10 μl / mg of tissue and cut into small fragments with scissors. Then the tissue / hydrolysis solution was subjected to ultrasound for 1 min at 6% duty cycle and was digested overnight at 37 ° C. After cleavage, the sample was centrifuged at a speed of 15,000 rpm and the tissue homogenate was aspirated and stored at 4 ° C.

The concentration of fluorescent compounds in tissues was determined on standard fluorescence curves obtained by adding known concentrations of introduced compounds to serum and tissue homogenates (in various dilutions) of control animals that were not injected with compounds. For each compound, a linear spacing was determined for each tissue. Fluorescence measurements were carried out using either a fluorescent plate reader or a fluorescence spectrometer. Tissue biodistribution results for molecules for selective delivery of 1, 2 and b are shown in FIG. 5. The unexpected result was that the distribution of the molecule for the selective delivery of b in the tumor tissue is 5 times higher compared to the molecules for the selective delivery of 1 and 2. This unexpected result is due to an asymmetric core consisting of an unequal number of positively and negatively charged amino acids of the peptide skeleton . Molecules for the selective delivery of 1 and 2 have an equal number of positively and negatively charged cores; as a result, the core is virtually neutral, while the molecule for selective delivery of b has a total charge of 3+ due to positively charged arginine residues. This shows that compounds with different numbers of acidic and basic amino acids have improved and useful properties ΐη νΐνο and have better and more useful biodistribution in comparison with symmetric molecules.

Example 17. S νΐνο detection of cancer metastases in the lymph nodes using PEET δΙ) \ 1.

Fluorescence labeling of metastatic cervical metastatic nodes after intravenous and peritumoral administration of fluorescent δΙ) \ 1 mice bearing a tumor.

The following model and analyzes were used to determine the ability of fluorescent δΙ) \ 1 to detect cancer metastases in the lymph nodes of immunocompetent bAb / c mice (Syag1e§ [Dueg, \\ 'ί1ηιίιψΙοη, MA 01887) carrying syngeneic ear tumors.

Mouse model. Mice, in groups of 4 mice, were placed in individually-ventilated TUS single-use cells (ΊηηοντνΌ, Шс., Δαη I Hashem CA 92121) and provided them with free access to standard laboratory food (Ca !. # 2018, Nag1ap ^ abο ^ aίο ^^ eδ, Sch. Iηά ^ аηарο1 ^ δ, Ш 4б250) and drinking water. Animals were kept under environmental conditions (12-hour daylight) for at least 5 days before implantation of tumor cells. All experiments were carried out in accordance with protocol # ЕВ11-002-009А, approved by КАСиС. Murine 4T1 tumor cells (ATCC® HitBG: CKB-2539 ™) and breast cancer (M1bb1e T 8119 subclone RUMT 8119) from the American Type Culture Collection (ATCC, Mañaδδaδ, UA 20108) and from the University of San Diego California ^ ap ϋίχ'μο, Sai & gsha) (υСδ ^, bа ΙοΠθ, CA 92093), respectively, were grown separately separately using standard methods of culturing cells. Tumor cells ( ⁴ × ^{10 5} tumor cells / 50 μl / mouse) were suspended in DZFR / Μaΐ ^^ de1 ™ (1: 1 vol.) And injected subcutaneously into the mouse’s auricle in the region above the cartilage to induce the growth of the primary tumor. Ιη νίνο imaging of the metastatic cervical lymph nodes of mice bearing an ear tumor was used to monitor the tumor of the mice as a surrogate mouse model of metastatic breast cancer that appeared seventeen to twenty days after tumor cell implantation.

Introduction 8ΌΜ test compound. For intravenous administration (injection into the tail vein), 8ΌΜ the movement of the mice was restricted using a rotating tail injector (CaP # KT1, Vashashee 8shpys, 1ps., Vasheye, ΜΑ 02185), and the test product (5-120 μM; 100 μl / mouse) was injected mice with a 28Ο ^1/2 insulin syringe (Ca !. # 14-826-79, WesUn.Ό ^ Ηηδοη apb Sátape, Rgapkyp Lakez, N1 07417). To carry out a peritumoral injection of 8 каждуюδ, each mouse participating in the test was anesthetized with a mixture of ketamine / xelazine (Ke1a | ec1® ^: & Xu1a__) ec1®, ΡΙκκίιιχ ΡΙκιγtaseiisa1z, 8ΐ. User,, 64506), administered intraperitoneally, and the test article (5-120 μM; 30-60 μl / ear) was injected subcutaneously around (around) the primary tumor and into the auricle of the opposite ear using a 300 Ρι ^^^ ΟΗ ^ ™ needle (Ca !. # 305106, Wesn ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^) After dosing, each mouse was returned to its intended cell and kept in a controlled environment until visualization. Fluorescence visualization of cervical lymph nodes was performed 1-24 hours after administration of the compound according to the procedure described below.

Fluorescence imaging. Deep anesthesia was used to visualize the cervical lymph nodes, injecting a ketamine / xylazine mixture intraperitoneally into each mouse. The mouse in a state of deep anesthesia was transferred to a sheet of black cork agglomerate (4x4 inches, Oiae! ®, ACCO Vgapbz, Γί ^ οΙ ^ 1, 1 600600, I8A) for blunt separation and imaging of cervical lymph nodes using a computerized fluorescent stereomicroscope 10, 8 ( , СО, ЦГО, бап), equipped with appropriate fluorescence filters both for detecting a single fluorescence intensity and for determining the fluorescence ratio of two fluorophores. For example, filters for Su5 and Su7 were used to determine the pairs of Su5 and Su7 based on PbAET 8ΌΜ with the participation of PbAET. After fluorescence imaging (see visualization of the ratio method below), the cervical lymph nodes were surgically removed, fixed in 10% buffered formalin and processed for histology (hematoxylin and eosin staining, H&E) to evaluate the correlation of fluorescence / cancer and determine the diagnostic characteristics 8ΌΜ.

A method for visualizing the ratio (intensity) of emission (radiation). Fluorescence images were obtained using an O1utris 8ΖΧ10 Kezeag 81όιόο ΥΙιοιόсюure research stereomicroscope (Otutseps Ateps, Sesheg Va11eu, ΡΑ). In the case of Su5 and Su7 RKET 8ΌΜ, to obtain two images at different radiation (emission) wavelengths, an excitation filter centered at 620 nm was used (Sygota ET620 / 60x, Sygota TesyηοοοООФ. Ве11ο ^ з Pa11з, УТ), and emission filters centered at 700 nm and at 810 nm (Sigot filters ET700 / 75t and ET810 / 90t). Images were obtained using the Ogsa-K2 camera (Natata! Zi, VpbdehuaYug, N6), connected to a computer based on the database. Two methods were used to determine the emission (emission) ratios in the case of lymph nodes. In one method, the intensity was averaged in the region of interest (KO1), including the portion of the lymph node of interest, or the entire lymph node of interest. Then, the ratio (intensities) of the emission (radiation) was calculated based on the intensities for each region of interest.

emission ratio Κοί = (ΓθίΙηΐ1 / Εχρ1) / (Ιηΐ2 / Εχρ2) (equation 1), where gOnH = average intensity for KO1 at the emission (emission) wavelength of 1 with an ET700 / 75t filter,

Exp1 = exposure time for 1n, gOn12 = average intensity for KO1 at an emission (emission) wavelength of 2 with an ET810 / 90t filter,

Exp2 = exposure time for 1p! 2.

The second method used to determine the emission ratio was based on averaging the emission ratio in the region of interest (KO1), including the part of the lymph node or the entire lymph node of interest obtained based on the ratio of emission signals. The ratio of the emission signals was obtained using a modified equation 1, including a scale factor, so that the pixel values were between 0 and 255 for an 8-bit image.

Emission ratio Рх = к * (ρχΙηΐ1 / Εχρ1) / (ρχΙηΐ2 / Εχρ2) (equation 2), where k = scale factor, рх1пН = intensity in pixels at the emission (emission) wavelength of 1 with an ET700 / 75t filter,

Exp1 = exposure time for 1pN,

- 56 030795 ρχΙηΐ2 = intensity in pixels at an emission (emission) wavelength of 2 with an ET810 / 90t filter,

Exp2 = exposure time for Ιηΐ2.

When determining the ratio (intensity) of emission for lymph nodes by both methods, quantitatively similar results were obtained.

The state of the lymph nodes, whether they are metastatic or non-metastatic, was determined by a pathomorphologist based on staining with H&E. Then, the contrast of the emission ratios for each δϋΜ (molecules for selective delivery) was quantified, dividing the average emission ratio of metastatic lymph nodes by the average emission ratio of non-metastatic lymph nodes and subtracting unity, as shown in equation 3

CEN - Me1AU / SopAU - 1 (Equation 3), where CEN = emission ratio contrast,

ΜеίАУ = average metastatic lymph node emission ratio,

SopAU = mean ratio of non-metastatic lymph node emission.

An example image of the emission ratio is shown in FIG. 6. The right picture shows a snapshot (image) of the relationship, which shows a high contrast between the metastatic lymph node (a very large node, shown by the dark arrow at the bottom left) and non-metastatic nodes (shown by other arrows). A higher ratio is shown as lighter pixels (metastatic node) compared to darker pixels with a lower ratio for non-metastatic nodes.

A contrast of 20 to 50% applicable to detecting cancer lymph nodes was considered good, an increase of 50 to 100% was considered high, while an increase above 100% was considered as a very high contrast.

table 2

Data for δϋΜ ΐη νίνο contrast ratios obtained on a mouse tumor model 4T1

Molecule for selective delivery IV Maximum contrast (low <20%, good 20% 50%, high> 50% - 100%, very high> 100%) Peritumoral maximum contrast (low <20%, good 20% - 50%, high> 50% - 100%, very high> 100%) 8ΌΜ-9 ηά Low 8ΌΜ-11 ηά Low 8ΌΜ-12 ηά Good 8ΌΜ-13 Good Good 8ΌΜ-14 Good Very tall 8ΌΜ-19 Good 3ΌΜ-20 Low 8ΌΜ-21 ηά Good 8ΌΜ-22 Low 8ΌΜ-23 Tall Very tall 8ΌΜ-24 Very tall Very tall 8ΌΜ-25 Very tall Very tall 8ΌΜ-27 Very tall ηά 8ΌΜ-28 Good Tall 8ΌΜ -29 ηά Tall 3ΌΜ-30 ηά Very tall 8ΌΜ-31 Low ηά 8ΌΜ-32 Very tall Very tall 8ΌΜ-33 Low Tall 8ΌΜ-35 Very tall Good 8ΌΜ-36 Tall Good 8ΌΜ-37 Low ηά δϋΜ-38 Good ηά 8ΌΜ-39 Good Tall 8ϋΜ-40 ηά Tall

pb = not determined

Example 18. Ex νίνο analysis of activity in relation to a murine tumor 8уΜТ 8119 tumor: splitting 57 030795 8EM and reaction ratios of RKET emission in mouse cancerous tissue compared to healthy tissue.

Samples of the tumor and muscle tissue of tumor-bearing mice RuMT 8119 were collected and frozen at -80 ° C. Tissues were thawed and homogenized in cold TSGOV buffer (pH 7.5, 50 mM TG18-HC1, 10 mM SaS1 _2, 150 mM № · ιθ Vgu35 and 0.05%) at 100 mg / 200 l, destroying sonication (VSX500, 8ots§ Ric & MaYupaE ., No. \\ 1o \ up. ST). Homogenates were centrifuged at a speed of 15,000 rpm at 4 ° C for 20 minutes, supernatants were collected. APMA (p-aminophenylmercury acetate, 90 μl, 2 mM in TCNB buffer) was added to supernatants (90 μl). Before use, the resulting mixture was incubated at 37 ° C for 1 h. 500 nM 8EM-23 (final volume: 50 μl) was used to cleave 45 μl of activated tissue supernatants. The analysis was performed on an 8res1gaMax M2 spectrometer using the 8th MaxRgo ν4.5 program. Fluorescence signals (Heh, 620 nm, Hat, 670 nm), (Heh, 620 nm, Hat, 773 nm) and (Heh, 720 nm; Hat, 773 nm), where Heh and Hat mean the wavelength of excitation and emission (radiation ), respectively, was determined at room temperature as a function of time. The samples were analyzed in triplicate, and the result of RKET 8EM splitting was an increased ratio of Su5 / Su7 fluorescence emission under experimental conditions: signals were recorded Su5 (Heh, 620 nm, Hat, 670 nm) and Su7 (Heh, 620 nm, Hat, 773 nm) .

Enzymatic activity when using tissues caused the cleavage of 8EM-23 and gave a large increase in the emission ratio of PKET (indicated primary tumor), as shown in FIG. 7. The increase in the ratio is the result of splitting 8EM. These data indicate that the 8EM-23 molecule is very active in the cancerous tissues of the breast tumor in mice and is cleaved significantly more in the cancerous tissue compared to normal muscle tissue, which is not active in this assay.

Example 19. Ex νί \ Ό analysis of human tissues: 8EM cleavage and the reaction of the ratio of RKET emission in human cancer tissue compared with healthy tissue.

Samples of tissue of a cancerous tumor of the human breast and healthy tissue of the human breast (provided by Sapseg Nitap T188ie No. 1 \\ όγ1 <) were homogenized in cold TCNB buffer (pH 7.5, 50 mM, Tp8-HC1, 10 mM CaCl ₂ , 150 mM ΝηΟ and 0.05% Vgu35) with a concentration of 100 mg / 200 μl using ultrasound (VCX500, §opu8 & Ma1epaE Shs, Kenti, ST). Homogenates were centrifuged at a speed of 15,000 rpm at 4 ° C for 20 minutes, supernatants were collected. Unless otherwise specified, 500 nM 8EM-23 (final volume: 50 μl) was used to cleave 45 μl of activated tissue supernatants. The analysis was performed on an 8res1gaMax M2 spectrometer using the 8th MaxRgo ν4.5 program. Fluorescence signals (Heh, 620 nm, Hat, 670 nm), (Heh, 620 nm, Hat, 773 nm) and (Heh, 720 nm; Hat, 773 nm), where Heh and Hat mean the wavelength of excitation and emission (radiation ), respectively, was determined at room temperature as a function of time. The samples were analyzed in triplicate, and the result of RKET 8EM splitting was an increased ratio of Su5 / Su7 fluorescence emission under experimental conditions: signals were recorded Su5 (Heh, 620 nm, Hat, 670 nm) and Su7 (Heh, 620 nm, Hat, 773 nm) . An example using 8EM-25 is shown in FIG. 8. Experiments with other 8EM5 were carried out according to the same procedure. The cleavage-dependent change in fluorescence can also be quantified as the rate of cleavage, the ratio of change per unit time, as shown in FIG. 9 for 8EM-25 and 8EM-32. Speeds were calculated based on the slope of the curve constructed from data obtained from 0 to 300 minutes.

Example 20. High sensitivity and specificity of the diagnostic method using 8EM on a model of metastatic lymph nodes.

The main indicators of the effectiveness of a diagnostic agent are sensitivity and specificity. Sensitivity reflects the ability to correctly recognize positive responses to a test. While specificity refers to the ability to correctly recognize adverse reactions to a test.

As an example of the high diagnostic efficiency using RET 8EM, we present the results obtained using 8EM-24 in a mouse model with a 4T1 metastatic tumor of the lymph nodes. δ EM-24 was administered by IV injection into the tail vein. From 3 to 6 hours after that, images of the lymph nodes of the mouse were obtained using the visualization of the fluorescence ratio described above to determine whether this ratio in the image of the lymph node is high (defined as a positive lymph node) or low (negative lymph node). Sensitivity and specificity were determined on the basis of a graph of the dependence of sensitivity on the frequency of false-positive conclusions (CBS), or CBS curves of the OShRS / EpluPlResha.ogdLuPl / Kesep'eg oregaypd skagasYugMyu). For analysis using CBS curves, the results were classified on a binary scale, positive and negative, taking into account the threshold value of the emission ratio. A CBS curve was constructed between the share of truly positive cases among positive results (level of truly positive results) and the share of false positive cases among negative results (level of false positives).

- 58 030795

True positive, false positive, truly negative and false negative results were determined by comparing the prognosis based on the data on the ratio of fluorescence emission and the threshold value with reference to positive or negative made by a pathomorphologist based on H&E staining. Emission ratio values for positive and negative results (as determined by H&E histopathology) are shown in FIG. 10. The threshold value was gradually adjusted from low to high, obtaining a complete K0C curve from (1, 1) or completely positive to (0, 0) or completely negative results. The K0C curve is shown in FIG. 11. To build this curve used data obtained in the study of 48 lymph nodes. It should be noted that sensitivity and specificity can be determined for each point on the K0Skrivoy. Sensitivity is the share (level) of truly positive results, while specificity matters one minus the share of false positive results (false positive level). The equations used to construct the K0C curve are shown below.

ΤΡΚ = TP / (TP + P ^ PPK = PP / (PP + T ^ where ΊΓΚ = share of truly positive results,

RRP = false positive rate,

TP = the number of truly positive results,

ΤN = number of true negative results,

PP = number of false positives,

ΡN = number of false negative results.

In this example, both sensitivity and specificity are 100% for all threshold values between 5.65 and 7.15. This means that the lymph nodes were correctly identified by the emission ratio method ΡΚΡΤ compared to the gold standard for the diagnosis of histopathological examination. Generally, for sensitivity and specificity, values> 90% are considered very high.

Example 21. The use of 8ΌΜ to visualize cancer in patients with breast cancer.

8ΌΜ-25 was intravenously administered to a breast cancer patient. Fluorescent particles at 8цент-25 were captured by cancer cells and / or tissue after cleavage of the linker. The light source was directed to the target tissue. Fluorescent particles emitted light, which was recorded using a camera or detector. The results obtained with a camera or detector were processed to obtain an image that allowed the surgeon to visualize cancer cells or tissue. The surgeon excised the indicated tissue for biopsy.

Example 22. The use of 8ΌΜ to visualize cancer in patients with prostate cancer.

8ΌΜ-26 was administered intravenously to a patient with prostate cancer. The fluorescent particles in 8ΌΜ-26 were captured by cancer cells and / or tissue after cleavage of the linker. The light source was directed to the target tissue. Fluorescent particles emitted light, which was recorded using a camera or detector. The results obtained with a camera or detector were processed to obtain an image that allowed the surgeon to visualize cancer cells or tissue. The surgeon excised the indicated tissue for biopsy.

Example 23. The use of 8ΌΜ to visualize cancer in patients with head and neck cancer (squamous cell carcinoma).

8ΌΜ-27 was administered intravenously to a patient with head and neck cancer. Fluorescent particles at 8ΌΜ-27 were captured by cancer cells and / or tissue after cleavage of the linker. The light source was directed to the target tissue. Fluorescent particles emitted light, which was recorded using a camera or detector. The results obtained with a camera or detector were processed to obtain an image that allowed the surgeon to visualize cancer cells or tissue. The surgeon excised the indicated tissue for biopsy.

Example 24. The use of 8ΌΜ to visualize cancer in patients with melanoma.

8ΌΜ-24 was intravenously administered to a patient with melanoma. Fluorescent particles at 8ΌΜ-24 were captured by cancer cells and / or tissue after cleavage of the linker. The light source was directed to the target tissue. Fluorescent particles emitted light, which was recorded using a camera or detector. The results obtained with a camera or detector were processed to obtain an image that allowed the surgeon to visualize cancer cells or tissue. The surgeon excised the indicated tissue for biopsy.

Example 25. The use of 8ΌΜ for imaging cancer in patients with thyroid cancer.

8ΌΜ-32 was administered intravenously to a thyroid cancer patient. Fluorescent particles at 8цент-32 were captured by cancer cells and / or tissue after cleavage of the linker. The light source was directed to the target tissue. Fluorescent particles emitted light, which was recorded using a camera or detector. The results obtained with a camera or detector were processed to obtain an image that allowed the surgeon to visualize cancer cells or tissue. The surgeon excised the indicated tissue for biopsy.

- 59 030795

LIST OF SEQUENCES <110> AVELAS BIOSAINSISC, INC.

<120> MOLECULES FOR SELECTIVE DELIVERY AND METHODS OF THEIR DELIVERY <130> 39088-708.601 <140> PCT / IB12 / 48732 <141> 2012-07-27 <150> 61 / 513,287 <151> 2011-07-29 <160> 40

<223> Failure to do: £ Arb1 £ 1c1a1 Bedieps: Bieberfeld <220>

<221> MOB_KEB <222> (4) .. (4) <223> Suz-Me <400> 1

Proof of Lei O1y Suz A1a O1y 1 5 <210> 2 <211> 6 <212> RCT <213> Arb1 £ 1s1a1

<223> Failure to do: £ Arb1 £ 1c1a1 Bedieps: Boolean less than <400> 2

Rgo Ley O1u Ley A1a O1u

5 <210> 3 <211> 9 <212> RCT <213> Arb1 £ 1s1a1 Bedieps <220>

<223> Failure to do: £ Arb1 £ 1c1a1 Bedieps: Bieberfeld <220>

- 60 030795 <221> t1zs_EeaEige <222> (1) .. (9) <223> YOU zediepe tau epotrazz 5 og 9 <400> 3

O1 and O1 and O1 and O1 and O1 and O1 and O1 and O1 and O1 and

5 hezSiez <210> 4 <211> 9 <212> RKT <213> ArE1E1s1a1 Zediepse <220>

<223> Weszr1pE1op about £ ArE1E1c1a1

ZUPEEETS <220>

<221> t1zs_EeaEige <222> (1) .. (9) <223> from zediepse tau epotrazz 8 oc 9 <400> 4

Agd Agd Agd Agd Agd Agd Agd Agd Agd 1 5 GezShiez <210> 5 <211> 5 <212> RCT <213> ArE1E1s1a1 Zedieps <220>

<223> Weszr1pE1op o £ ArE1E1c1a1 Zedieps

O1 and O1 and O1 and O1 and O1 and

5

ZUPEEETS <210> 6 <211> 8 <212> RCT <213> ArE1E1s1a1 Zediepse <220>

<223> Weszr1pE1op o £ ArE1E1c1a1 Zediepse reE1Ne <400> 6

Agd Agd Agd Agd Agd Agd Agd Agd Agd

5

ZUPEeets <210> 7 <211> 8 <212> RCT <213> ArE1E1s1a1 Zediepse <220>

- 61 030795

<223> Vezsg1rё1op o £ ΑΓύί £ ί ^ α1 Zediepse: Dumbbells rer + fb <400> 7 Α ^ Rgo ей Α α1α ей Т Tgr Α ^ Zeg one 5 <210> 8 <211> 8 <212> Rkt <213> ΑΓύί £ ί ^ α1: Zediepse <220> <223> Vessg1rё1op o £ ΑΓύί £ ίοί & 1 Zediepse: Dumbbells rer + fb <400> 8

O1 and Zeg Rgo A1a Tug Tug Tg A1a 1 5 <210> 9 <211> 6 <212> RCT <213> ΑΓύίίίοίαΙ Zediepse <220>

<223> Vezsg1rё1op o £ ΑΓύίίίοίαΙ Zediepse: Zup + bе + Fs rer + Фее <400> 9

Azr Rgo Agd Zeg Rybey

5 <210> 10 <211> 6 <212> RCT <213> ΑΓύίίίοίαΙ Zediepse <220>

<223> Vezsg1rё1op o £ ΑΓύίίίοίαΙ Zedieppes: Zup + bе + Fs repr + Фее <400> 10

Рgo Рго Α ^ Зег Ръе Бей

5 <210> 11 <211> 6 <212> RCT <213> ΑΓύίίί ^ αΙ Zediepse <220>

<223> Weszr1pO1op o £ ΑΓύίίί ^ αΙ Zediepps: ZnBeSeRs + PhBe <400> 11

Α ^ ей О О 1 уз уз уз уз уз

5

- 62 030795 <210> 12 <211> 5 <212> RKT <213> ArE1 £ 1s1a1 Zediepse <220>

<223> LESG1RE1OP about £ ArE1 £ 1c1a1 Zedieppes: ZUPEeE1s with the rest <220>

<221> M0_KEZ <222> (5) .. (5) <223> Luz-As <400> 12

Agd Ley Ojn Ley Luz

one 5 <210> 13 <211> 9 <212> Rkt <213> ArE1 £ 1s1a1 Zediepse <220> <223> Ез с г 1 р о о о £ Аг ArE1 1 1c1a1 Zediepse: ZUPEEEE1s reE1be <400> 13 O1 and O1 and C1 and O1 and C1 and O1 and O1 and C1 and O1i one 5

<210> fourteen <211> 9 <212> Rkt <213> ArE1 £ 1s1a1 Zediepse <220> <223> Bzr1pE1op o £ ArE1 £ 1c1a1 Zediepse: ZUPEEEE1s <400> fourteen Agd Agd Agd Agd Agd Agd Agd Agd Agd Agd one 5

<210> 15 <211> 20 <212> RCT <213> ArE1 £ 1s1a1 Zediepse <220>

<223> LESG1RE1OP about £ ArE1 £ 1c1a1 Zedieppes: ZUPEeE1s with the rest <220>

<221> t1zs_ £ eaEige <222> (1) .. (20) <223> from zediepece tau epotrazz 2-20

- 63 030 795 <400> 15

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5 10 15

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<223> Weszr1p51op o £ Ar51 £ 1s1a1

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one 5 10 fifteen O1i O1 and O1 and O1i twenty

<210> 17 <211> 8 <212> Rkt <213> Ag51 £ 1s1a1 Zediepse <220> <223> Weszr1p51op o £ Ar51 £ 1s1a1

<220> <221> t1zs_ £ ea5ige epotrazz 5-8 ges15. <222> <223> (1) .. (8) YOU zedieps tau <400> 17 O1 and O1 and O1 and O1 and O1 and O1 and O1 and O1 and one 5

<210> 18 <211> 7 <212> RCT <213> Ar51 £ 1s1a1 Zediepse <220>

<223> Weszr1p51op o £ Ar51 £ 1s1a1

- 64 030 795 <220>

<221> t1zs_ £ eakige <222> (1) .. (7) <223> from zediepece tau epotrazz 5-7 dzhezsiez <400> 18

O1 and O1 and O1 and O1 and O1 and O1 and O1 and

5 <210> 19 <211> 6 <212> RCT <213> Lgk1 £ 1s1a1 Zediepse <220>

<223> Weszgrk1op o £ Ark1 £ 1s1a1 Zediepse: 5kbkeksrrk10.е <400> 19

O1 and O1 and O1 and O1 and O1 and O1 and

5 <210> 20 <211> 7 <212> RCT <213> Lgk1 £ 1s1a1 Zediepse <220>

<223> Weszgrk1op o £ Ark1 £ 1s1a1 Zedieppes: 5kbkeks with rek10.е <400> 20

O1 and O1 and O1 and O1 and O1 and O1 and O1 and

5 <210> 21 <211> 8 <212> RCT <213> Lgk1 £ 1s1a1 Zediepse <220>

<223> Weszgrk1op o £ Ark1 £ 1s1a1 Zediepse: 5kbkeks with rek10.е <400> 21

O1 and O1 and O1 and O1 and O1 and O1 and O1 and O1 and

5 <210> 22 <211> 20 <212> RCT <213> Lgk1 £ 1s1a1 Zediepse <220>

<223> Weszkrk1op o £ Ark1 £ 1s1a1 Zedieps: 5kkkk1s rek10.е

- 65,030,795 <220>

<221> T1ZS_DeaDige <222> (1) .. (20) <223> From zediepse tau epotrazz 5-20 gezSiez <400> 22

Agd Agd Agd Agd Agd Agd Agd Agd Agd Agd Agd Agd Agd Agd Agd Agd Agd 1 5 10 15

Agd Agd Agd Agd 20 <210> 23 <211> 12 <212> RCT <213> AgD1D1s1a1 Zediepse <220>

<223> Weszr1rD1op o £ ArD1D1s1a1 Zedieps: ZupDieDDs reResche <220>

<221> T1ZS_DeaDige <222> (1) .. (12) <223> From zediepse tau epotrazz 5-12 gezSiez <400> 23

Agd Agd Agd Agd Agd Agd Agd Agd Agd Agd Agd Agd 1 5 10 <210> 24 <211> 9 <212> RCT <213> AgD1D1s1a1 Zedieppes <220>

<223> VezsgDrDDop oD AgD1D1s1a1 Zediepse: ZupDieDDs regReshe <220>

<221> T1ZS_DeaDige <222> (1) .. (9) <223> From zediepse tau epotrazz 7-9 hezSiez <400> 24

Agd Agd Agd Agd Agd Agd Agd Agd Agd Agd

5 <210> 25 <211> 8 <212> RCT <213> ArD1D1s1a1 Zedieppes <220>

<223> VezsgDrDDop oD AgD1D1s1a1 Zediepse: ZupDieDDs regReshe

- 66 030795 <220>

<221> t1zs_ £ eaёige <222> (1) .. (8) <223> from zediepse tau epotrazz 7-8 gezSiez <400> 25

Agd Agd Agd Agd Agd Agd Agd Agd Agd

5 <210> 26 <211> 7 <212> RCT <213> ArE1 £ 1s1a1 Bedieps <220>

<223> Weszr1pO1op about £ ArE1 £ 1c1a1 Bedieps: The recession is <400> 26

Agd Agd Agd Agd Agd Agd Agd

5 <210> 27 <211> 6 <212> RCT <213> ArE1 £ 1s1a1 Bedieps <220>

<223> Weszr1pO1op about £ ArE1 £ 1c1a1 Bedieps: A recessionary book <220>

<221> MOB_KEB <222> (4) .. (4) <223> Apu albpo as1D <400> 27

ProofLei O1y Haa A1a O1y 1 5 <210> 28 <211> 10 <212> RCT <213> ArE1 £ 1s1a1 Bedieps <220>

<223> Weszr1pE1op o £ ArE1 £ 1c1a1 Bedieps: BieberRepre10.e <400> 28

O1u O1u A1a A1a Azp Ley Wa1 Agd O1u O1u

5 10 <210> 29 <211> 10 <212> RCT <213> ArE1 £ 1s1a1 Bedieps

- 67 030795

<220> <223> Zediepse: Zupneb1s PEP1Ee <400> 29th Zeg O1u Agd 11e O1u Rne Ley Agd TNG A1a one 5 10 <210> thirty <211> 5 <212> Rkt <213> Ar1lc1a1 Zediepse <220> <223> Ез с г 1 р оп оп о о Аг Zediepse: Zooper PEP1Ee <400> thirty

Zeg O1y Agd Zeg A1a 1 5 <210> 31 <211> 4 <212> RCT <213> Arb1yc1a1 Zediepse <220>

<223> bzb1pb1o0 £ Arb1lc1a1 Zediepse: Zlbieb1c pbb15.e <400> 31

О1у РЬе Лей О1у <210> 32 <211> 4 <212> RCT <213> Arb1yc1a1 Zedieps <220>

<223> ез с г 1 р оп о о £ Аг Ь Ь 1 1 Ы 1 З З З ед ие ие:: З З уп уп Ь Ь с с Ь Э Э Э 400 400 <<

A1a Lea A1a Lea <210> 33 <211> 5 <212> RCT <213> Arb1lc1a1 Zedieps <220>

<223> ез ез г 1 р оп о о Аг Аг Аг Аг 1 1 Ы З З З З З З ие:: З З:: Ь Ь с с <<<<<<220>

- 68 030795

<221> M0_KEZ <222> (3) .. (3) <223> ZEBU1-Suz <400> 33 Рgo 11е Souz РЬе РЬе one 5

<210> 34 <211> 8 <212> RKT <213> ArE1 £ 1s1a1 Zediepse <220> <223> Ledzelpreop about £ ArE1 £ 1s1a1 <400> 34 O1u O1u Rgo Agd O1u Ley Rgo O1u one 5

<210> 35 <211> 6 <212> Rkt <213> ArE1 £ 1s1a1 Zediepse <220> <223> Ledzelpreop about £ ArE1 £ 1s1a1 <400> 35 NTZ Zeg Zeg Luz Ley O1n one 5

<210> 36 <211> 10 <212> Rkt <213> ArE1 £ 1s1a1 Zediepse <220> <223> Bzr1pE1op o £ ArE1 £ 1c1a1 Zediepse: ZUPEEEE1s <400> 36 Еи а а 1 еи еи А а а а ег O1u Tug one 5 10

<210> 37 <211> 10 <212> Rkt <213> ArE1 £ 1s1a1 Zediepse <220> <223> Bzr1pE1op o £ ArE1 £ 1c1a1 Zediepse: ZUPEEEE1s <400> 37 O1y Wa1 Zeg O1p Azp Tug Rgo 11e Wa1 O1u one 5 10

- 69 030795

<210> 38 <211> 10 <212> Rkt <213> AgB1 £ 1s1a1 Zediepse <220> <223> LezgbrbrBbop o £ ArB1 £ 1s1a1 reBbDe Zediepse: ZupBieBbs <400> 38 O1y Va1 Va1 O1p A1a Zeg Suz Agd Ley A1a one 5 10

<210> 39 <211> 4 <212> RCT <213> ArB1 £ 1s1a1 Zediepse <220>

<223> LezgbrbrBbop o £ ArB1 £ 1s1a1 Zediepse: ZupBeBbs rerbbDe <400> 39

Azr O1 and Va1 Azr <210> 40 <211> 6 <212> RCT <213> ArB1 £ 1s1a1 Zedieps <220>

<223> LezgbrbrBbop o £ ArB1 £ 1s1a1 reBbDe Zediepse: ZupBeBbs <400> 40 O1u Tgr O1i Nbz Azr O1u fifteen <210> <211> <212> <213> 41 9 Rkt AgB1 £ 1s1a1 Zediepse <220> <223> LsgbrbrBbop o £ ArB1 £ 1s1a1 Zediepse: ZupBeBbs

rerbbde <220>

<221> T1ZS_ £ eaBige <222> (1) .. (9) <223> YOUR HEADSET TOW ENCLOSURE 5-9 GESBEEZE <400> 41

O1 and O1 and O1 and O1 and O1 and O1 and O1 and O1 and O1 and

5

- 70 030795

Claims (34)

CLAIM 1. The molecule of selective delivery of a pair of agents for imaging cancer tissue, having the formula I, having the structure [Ώα-ca] - A- [cm-M] -X-B- [sv-ϋΒ] formula I, where X is a peptide linker which is capable of being cleaved by a protease; A is a peptide with a sequence containing 5 consecutive glutamate residues; B is a peptide with a sequence containing 8 consecutive arginine residues; c _A , c _B , and ο _Μ each independently represent an amino acid residue; M is a polyethylene glycol polymer (PEG); Ed and Ό _Β are a pair of acceptor and donor fluorescent particles between which fluorescence resonant energy transfer by the Förster mechanism is possible; and | ρ ·, | -Μ | attached in any position to X, [And _A -c _A ] attached to any amino acid in A, [c _B E _B | attached to any amino acid in B; Μ is a polyethylene glycol polymer having an average molecular weight of 500, 1000, 2000, 5000 or 10000 Da. 2. The molecule according to claim 1, in which with _A , _B and ο _Μ , each independently selected from Ό-cysteine, Ό glutamate, lysine and para-4-acetyl-E-phenylalanine. 3. The molecule of claim 1, wherein _B represents a residue Ό-cysteine. 4. The molecule according to claim 1, in which with _A represents the remainder of Ό-glutamate or lysine. 5. The molecule according to claim 1, in which ο _Μ denotes the remainder of para-4-acetyl-E-phenylalanine. 6. The molecule according to claim 1, in which X is capable of cleavage using matrix metalloproteinase. 7. The molecule according to claim 1, in which X contains an amino acid sequence that is capable of cleavage using ΜΜΡ2, ΜΜΡ7, ΜΜΡ9 or ΜΜΡ14. 8. The molecule according to claim 1, in which X contains an amino acid sequence selected from REOEAO, REO-C (Sche) -AO, KP1. L1LAK8, E8RAUTA, IRK8PE, PPK8PE, Kromk. and KEREK (Ac). 9. The molecule according to claim 1, in which X contains the amino acid sequence REOEAO. 10. The molecule according to claim 1, in which X contains the amino acid sequence of REO-C (sce) -AO. 11. The molecule according to claim 1, in which X contains the amino acid sequence KRTATAUK8. 12. The molecule according to claim 1, in which And _A and E _In mean Su5 and Su7. 13. The molecule according to claim 1, in which And _A and E _In mean Cy5 and IK ^ ye750. 14. The molecule according to claim 1, in which And _A and E _In mean Cy5 and IK ^ u800. 15. The molecule according to claim 1, in which both _A and E _B mean Su5 and! CO. 17. The molecule according to claim 1, wherein the tissue is breast cancer tissue, colorectal cancer tissue, squamous cell carcinoma tissue, prostate cancer tissue, melanoma tissue, or thyroid cancer tissue. 18. The molecule according to claim 1, wherein the tissue is breast cancer tissue. 19. The molecule according to claim 1, wherein the tissue is colorectal cancer tissue. 20. A tissue sample for imaging cancer, containing (a) tissue and (b) the molecule according to any one of paragraphs.116. 21. The tissue sample according to claim 20, which is a sample placed on a slide, or a slice to determine the pathology. 22. The tissue sample of claim 20, wherein the tissue is cancerous tissue. 23. The tissue sample of claim 20, wherein the cancerous tissue is breast cancerous tissue, colorectal cancerous tissue, squamous cell carcinoma tissue, prostate cancerous tissue, melanoma tissue or thyroid cancerous tissue. 24. The tissue sample of claim 20, wherein the cancerous tissue is breast cancerous tissue. - 71 030795 glands. 25. The tissue sample of claim 20, wherein the cancerous tissue is colorectal cancerous tissue. 26. The use of the molecule according to claim 1 for the delivery of a pair of imaging agents into the cancerous tissue, including bringing the cancerous tissue into contact with said molecule. 27. The use of claim 26, wherein the cancerous tissue is breast cancerous tissue, colorectal cancerous tissue, squamous cell carcinoma tissue, prostate cancerous tissue, melanoma tissue or thyroid cancerous tissue. 28. The use of claim 26, wherein the cancerous tissue is breast cancerous tissue. 29. The use of claim 26, wherein the cancerous tissue is cancerous tissue of colorectal cancer. 30. The use of a molecule according to any one of claims 1 to 16 for visualizing cancer tissue in a subject in need thereof, including: ι) the introduction of a specified molecule to the subject; and ΐΐ) visualization of at least one of the imaging agents. 31. The application of claim 30, wherein the cancerous tissue is breast cancerous tissue, colorectal cancerous tissue, squamous cell carcinoma tissue, prostate cancerous tissue, melanoma tissue or thyroid cancerous tissue. 32. The application of claim 30, wherein the cancerous tissue is breast cancerous tissue. 33. The application of clause 30, in which the cancerous tissue is a cancerous tissue of colorectal cancer. 34. The application of claim 30, wherein the surgical field surrounding the cancerous tissue is reduced. 35. The molecule of the formula 8ΌΜ-25 8ΌΜ-25. Sop1ta1a1eg1 ΐίδδΐιθ